Prodrug Comprising a Drug Linker Conjugate

ABSTRACT

The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof comprising a drug linker conjugate D-L, wherein -D is an amine containing biologically active moiety; and -L is a non-biologically active linker moiety -L 1  represented by formula (I): 
     
       
         
         
             
             
         
       
     
     wherein the dashed line indicates the attachment to the amine of the biologically active moiety and wherein R 1 , R 1a , R 2 , R 2a , R 3 , R 3a , X, X 1 , X 2 , X 3  have the meaning as indicated in the description and the claims and wherein L 1  is substituted with one to four groups L 2 -Z and optionally further substituted, provided that the hydrogen marked with the asterisk in formula (I) is not replaced by a substituent; wherein L 2  is a single chemical bond or a spacer; and Z is a carrier group. The invention also relates to A-L, wherein A is a leaving group, pharmaceutical composition comprising said prodrugs and their use as medicaments.

The present application is a continuation of U.S. patent applicationSer. No. 14/528,362 filed on Oct. 30, 2014, which is a continuation ofU.S. patent application Ser. No. 12/865,693, filed on Jul. 30, 2012,which is a national phase of and thus claims priority from, PCT PatentApplication No. PCT/EP2009/051079 filed on Jan. 30, 2009—the disclosuresof which are incorporated herein by reference in their entirety—whichclaims priority from EP 08170872.9 filed on Dec. 5, 2008, and from EP08150973.9 filed on Feb. 1, 2008.

The present invention relates to a prodrug or a pharmaceuticallyacceptable salt thereof comprising a drug linker conjugate D-L. Theinvention also relates to pharmaceutical compositions comprising saidprodrugs and their use as medicaments.

To enhance physicochemical or pharmacokinetic properties of a drug invivo such drug can be conjugated with a carrier.

Typically, carriers in drug delivery are either used in a non-covalentfashion, with the drug physicochemically formulated into asolvent-carrier mixture, or by covalent attachment of a carrier reagentto one of the drug's functional groups.

However the non-covalent approach requires a highly efficient drugencapsulation to prevent uncontrolled, burst-type release of the drug.Restraining the diffusion of an unbound, water soluble drug moleculerequires strong van der Waals contacts, frequently mediated throughhydrophobic moieties. Many conformationally sensitive drugs, such asproteins or peptides, are rendered dysfunctional during theencapsulation process and/or during subsequent storage of theencapsulated drug. In addition, such amino-containing drugs readilyundergo side reactions with carrier degradation products (see, forexample, D. H. Lee et al., J. Contr. Rel., 2003, 92, 291-299).Furthermore, dependence of the release mechanism of the drug uponbiodegradation may cause interpatient variability.

Alternatively, the drugs may be conjugated to a carrier through covalentbonds. This approach is applied to various classes of molecules, fromso-called small molecules, through natural products up to largerproteins. Covalent drug carrier conjugates can be divided into twogroups. Firstly, conjugates, where the covalent bond between carrier anddrug is mostly present during the action of the drug (“permanentcovalent bond”), i.e. a derivative of the drug exhibits itspharmacological effects as it is known for the drug as such. Secondly,the covalent bond is mostly previously cleaved to release the drug assuch, which can exhibit its known pharmacological effects. In the lattercase the covalent drug carrier conjugate is called carrier linkedprodrug or carrier prodrug.

In order to ensure cleavage of the covalent bond between carrier anddrug easy removal of said bond in vivo is required to release the drug(prodrug activation).

Prodrug activation may occur by enzymatic or non-enzymatic cleavage ofthe bond between the carrier and the drug molecule, or a sequentialcombination of both, i.e. an enzymatic step followed by a non-enzymaticrearrangement.

Enzymatically induced prodrug activation is characterized in that thecleavage in enzyme-free in-vitro environment such as an aqueous buffersolution, of, e.g., an ester or amide may occur, but the correspondingrate of hydrolysis may be much too slow and not therapeutically useful.In an in-vivo environment, esterases or amidases are typically presentand the esterases and amidases may cause significant catalyticacceleration of the kinetics of hydrolysis from twofold up to severalorders of magnitude. Therefore, the cleavage is predominantly controlledby the enzymatic reaction.

A major drawback of predominantly enzymatic cleavage is interpatientvariability. Enzyme levels may differ significantly between individualsresulting in biological variation of prodrug activation by the enzymaticcleavage. The enzyme levels may also vary depending on the site ofadministration. For instance it is known that in the case ofsubcutaneous injection, certain areas of the body yield more predictabletherapeutic effects than others. To reduce this unpredictable effect,non-enzymatic cleavage or intramolecular catalysis is of particularinterest.

Therefore, enzyme-independent autocatalytic cleavage of carrier andbiologically active moiety is preferred. In most cases this is achievedby an appropriately designed linker moiety between the carrier and thebiologically active moiety, which is directly attached to the functionalgroup of a biologically active moiety via covalent bond.

Specific linker types are known in the art.

Y. Sohma et al., J. Med. Chem. 46 (2003), 4124-4135 describe ester basedprodrugs, where the carrier is water-soluble and the biologically activemoiety is derived from HIV-1 protease inhibitor KNI-727. The linker usedis attached to the biologically active moiety via ester group. Themechanism of this prodrug system is cyclization-activation by cyclicimide formation for the cleavage of ester bonds.

However this is disadvantageous because of the instability of the esterfunctional group. Furthermore, ester groups may be less chemoselectivelyaddressable for the conjugation of the carrier or linker and the drug.

A. J. Garman et al. (A. J. Garman, S. B. Kalindjan, FEBS Lett. 1987, 223(2), 361-365 1987) use PEG5000-maleic anhydride for the reversiblemodification of amino groups in tissue-type plasminogen activator andurokinase. Regeneration of functional enzyme from PEG-uPA conjugate uponincubation at pH 7.4 buffer by cleavage of the maleamic acid linkagefollows first order kinetics with a half-life of 6.1 h. A disadvantageof the maleamic acid linkage is the lack of stability of the conjugateat lower pH values. This limits the applicability of the maleamic acidlinkage to biologically active agents which are stable at basic (high)pH values, as purification of the biologically active agent polymerconjugate has to be performed under basic (high pH) conditions toprevent premature prodrug cleavage.

In WO-A 2004/108070 prodrug system based onN,N-bis-(2-hydroxyethyl)glycine amide (bicine) linker is described. Inthis system two PEG carrier molecules are linked to a bicine moleculecoupled to an amino group of the drug molecule. The first two steps inprodrug activation is the enzymatic cleavage of the first linkagesconnecting both PEG carrier molecules with the hydroxy groups of thebicine activating group. Different linkages between PEG and bicine aredescribed resulting in different prodrug activation kinetics. The secondstep in prodrug activation is the cleavage of the second linkageconnecting the bicine activating group to the amino group of the drugmolecule. The main disadvantage of this system is the connection of thepolymer to the bicine linker resulting in a slow hydrolysis rate of thissecond bicine amide linkage (t½>3 h in phosphate buffer). Consequentlythe release of a bicine-modified prodrug intermediate may show differentpharmacokinetic, immunogenic, toxicological, and pharmacodynamicproperties as compared to the parent native drug molecule.

Another bicine-based system is described in WO-A 2006/136586.

Accordingly, there is a need for alternative carrier-linked prodrugs,where the linker allows an autocatalytic cleavage to release a drug inan unmodified form without remaining residues originating from thelinker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of the cleavage resulting in a cyclic imide.

FIGS. 2A-2G, collectively referred to as FIG. 2, depict further detailsconcerning compound numerals, starting materials, synthesis method,molecular weight (MW), and MS data.

FIG. 3 shows in vivo and in vitro linker cleavage data of 13b, whereinin vivo (triangles) and in vitro (diamonds) cleavage kinetics are shownby semilogarithmic representation.

DETAILED DESCRIPTION OF EMBODIMENTS

Thus, an object of the present invention is to provide such drug linkerconjugates, where the linker is covalently attached via a cleavable bondto a biologically active moiety (representing the drug after release),and where the linker is further covalently attached via a permanent bondto a carrier directly or via a spacer to form the carrier-linkedprodrug.

This object is achieved by a prodrug or a pharmaceutically acceptablesalt thereof comprising a drug linker conjugate D-L, wherein:

-D is a nitrogen containing biologically active moiety; and-L is a non-biologically active linker moiety L¹ represented by formula(I):

wherein the dashed line indicates the attachment to the nitrogen of thebiologically active moiety by forming an amide bond;X is C(R⁴R^(4a)), N(R⁴), O, C(R⁴R^(4a))—C(R⁵R^(5a)),C(R⁵R^(5a))—C(R⁴R^(4a)), C(R⁴R^(4a))—N(R⁶), N(R⁶)—C(R⁴R^(4a)),C(R⁴R^(4a))—O, or O—C(R⁴R^(4a));

X¹ is C; or S(O);

X² is C(R⁷, R^(7a)); or C(R⁷, R^(7a))—C(R⁸, R^(8a));

X³ is O; S; or N—CN;

R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵, R^(5a), R⁶, R⁷,R^(7a), R⁸, R^(8a) are independently selected from the group consistingof H; and C₁₋₄ alkyl;Optionally, one or more of the pairs R^(1a)/R^(4a), R^(1a)/R^(5a),R^(4a)/R^(5a), R^(7a)/R^(8a) form a chemical bond;Optionally, one or more of the pairs R¹/R^(1a), R²/R^(2a), R⁴/R^(4a),R⁵/R^(5a), R⁷/R^(7a), R⁸/R^(8a) are joined together with the atom towhich they are attached to form a C₃₋₇ cycloalkyl; or 4 to 7 memberedheterocyclyl;Optionally, R⁴/R⁶ are joined together with the atoms to which they areattached to form a saturated 4 to 7 membered heterocyclyl;Optionally, one or more of the pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵, R⁴/R⁶,R⁷/R⁸, R²/R³, are joined together with the atoms to which they areattached to form a ring A;Optionally, R³/R^(3a) are joined together with the nitrogen atom towhich they are attached to form a 4 to 7 membered heterocycle;A is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; and9 to 11 membered heterobicyclyl; andwherein L¹ is substituted with one to four groups L²-Z and optionallyfurther substituted, provided that the hydrogen marked with the asteriskin formula (I) is not replaced by a substituent; whereinL² is a single chemical bond or a spacer; andZ is a carrier group.

It was surprisingly found, that the scope of cyclization-activation bycyclic imide formation can be extended from ester to even carrier-linkedamide prodrugs, despite the much greater stability of the amide bondunder aqueous conditions. It was observed that N,N′-biscarboxarnideslinked to a nucleophile carrying moiety through one amide bond and tothe drug molecule through the second amide bond exhibit autohydrolysisin a range that is useful for prodrug applications. In addition, it wasdiscovered that linkers can be designed that include a carrierpermanently attached to the N,N′ biscarboxamide motif in such a fashionthat cyclic imide formation can be employed as a self-activationprinciple in carrier-linked amide prodrug design.

Examples for such preferred cyclic cleavage products are substitutedsuccinimide or glutarimide ring structures. Prerequisite for suchcyclization activation is the presence of an amine-containingnucleophile in the linker structure and another amide bond which is notthe amide prodrug bond but an amide bond substituted with a hydrogenatom.

In case of succinimide- or a glutarimide-activated prodrug cleavage, theamine-containing nucleophile serves as a neighbouring group to enhancethe nucleophilicity of the nitrogen contained in the permanent amidebond which in turn attacks the prodrug amide carbonyl group andconsequently induces intramolecular acylation of the permanent amidebond generating the cyclic imide ring.

Therefore preferred linker structures comprise a permanent linkage to acarrier, an amine-containing nucleophile, and a permanent amide bondwith a hydrogen attached to the nitrogen of the amide bond.Corresponding carrier-linked prodrugs comprise a linker containing apermanent linkage to a carrier, an amine-containing nucleophile and saidpermanent amide bond, and a nitrogen containing biologically activemoiety derived from the drug conjugated to the linker by means of acleavable amide bond.

FIG. 1 shows an example of the cleavage resulting in a cyclic imide. Thenitrogen of the biologically active moiety is shown as hydrogencontaining amine, which results in a drug having a primary aminefunctional group. However also, e.g., a secondary amine may be part ofthe drug. For simplification reasons the one to four mandatorysubstituents L²-Z including the carrier are not shown.

Preferred properties of the prodrug are given by a half-life ofhydrolysis in aqueous buffer at pH 7.4 and 37° C. between 1 h and 3months; similar rates of hydrolysis under physiological conditions inbuffer and plasma.

The prodrug according to the present invention may show excellent invivo/in vitro correlation of linker cleavage, a high degree of enzymeindependence and can be stored at lower pH (pH dependent cleavage).

Within the meaning of the present invention the terms are used asfollows.

“Biologically active moiety D” means the part of the drug linkerconjugate, which results after cleavage in a drug D-H of knownbiological activity.

“Non-active linker” means a linker which does not show thepharmacological effects of the drug derived from the biologically activeagent.

“Alkyl” means a straight-chain or branched carbon chain. Each hydrogenof an alkyl carbon may be replaced by a substituent.

“C₁₋₄ alkyl” means an alkyl chain having 1-4 carbon atoms, e.g. ifpresent at the end of a molecule: methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl tert-butyl, or e.g. —CH₂—, —CH₂—CH₂—,—CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of amolecule are linked by the alkyl group. Each hydrogen of a C₁₋₄ alkylcarbon may be replaced by a substituent.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—,—C(CH₃)₂—, when two moieties of a molecule are linked by the alkylgroup. Each hydrogen of a C₁₋₆ alkyl carbon may be replaced by asubstituent.

Accordingly, “C₁₋₁₈ alkyl” means an alkyl chain having 1 to 18 carbonatoms and “C₈₋₁₈ alkyl” means an alkyl chain having 8 to 18 carbonatoms. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chain having 1 to 50carbon atoms.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —CH═CH₂,—CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—,when two moieties of a molecule are linked by the alkenyl group. Eachhydrogen of a C₂₋₅₀ alkenyl carbon may be replaced by a substituent asfurther specified. Accordingly, the term “alkenyl” relates to a carbonchain with at least one carbon double bond. Optionally, one or moretriple bonds may occur.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms, e.g. if present at the end of a molecule: —C≡CH,—CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties ofa molecule are linked by the alkynyl group. Each hydrogen of a C₂₋₅₀alkynyl carbon may be replaced by a substituent as further specified.Accordingly, the term “alkynyl” relates to a carbon chain with at leastone carbon triple bond. Optionally, one or more double bonds may occur.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated, e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of acycloalkyl carbon may be replaced by a substituent. The term “C₃₋₇cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridged bicycleslike norbonane or norbonene. Accordingly, “C₃₋₅ cycloalkyl” means acycloalkyl having 3 to 5 carbon atoms.

Accordingly, “C₃₋₁₀ cycloalkyl” means a cyclic alkyl having 3 to 10carbon atoms, e.g. C₃₋₇ cycloalkyl; cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl. The term “C₃₋₁₀ cycloalkyl” also includes atleast partially saturated carbomono- and bicycles.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine.

“9 to 11 membered heterobicyclyl” or “9 to 11 membered heterobicycle”means a heterocyclic system of two rings with 9 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom. Examples for a 9 to 11membered heterobicycle are indole, indo line, benzofuran,benzothiophene, benzoxazole, benzisoxazole, benzothiazole,benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline,decahydroquinoline, isoquinoline, decahydroisoquinoline,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine orpteridine. The term 9 to 11 membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane.

In case the compounds according to formula (I) contain one or moreacidic or basic groups, the invention also comprises their correspondingpharmaceutically or toxicologically acceptable salts, in particulartheir pharmaceutically utilizable salts. Thus, the compounds of theformula (I) which contain acidic groups can be used according to theinvention, for example, as alkali metal salts, alkaline earth metalsalts or as ammonium salts. More precise examples of such salts includesodium salts, potassium salts, calcium salts, magnesium salts or saltswith ammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Compounds of the formula(I) which contain one or more basic groups, i.e. groups which can beprotonated, can be present and can be used according to the invention inthe form of their addition salts with inorganic or organic acids.Examples for suitable acids include hydrogen chloride, hydrogen bromide,phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, aceticacid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formicacid, propionic acid, pivalic acid, diethylacetic acid, malonic acid,succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid,isonicotinic acid, citric acid, adipic acid, and other acids known tothe person skilled in the art. If the compounds of the formula (I)simultaneously contain acidic and basic groups in the molecule, theinvention also includes, in addition to the salt forms mentioned, innersalts or betaines (zwitterions). The respective salts according to theformula (I) can be obtained by customary methods which are known to theperson skilled in the art like, for example by contacting these with anorganic or inorganic acid or base in a solvent or dispersant, or byanion exchange or cation exchange with other salts. The presentinvention also includes all salts of the compounds of the formula (I)which, owing to low physiological compatibility, are not directlysuitable for use in pharmaceuticals but which can be used, for example,as intermediates for chemical reactions or for the preparation ofpharmaceutically acceptable salts.

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMEA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, preferably in humans.

“Pharmaceutical composition” means one or more active ingredients, andone or more inert ingredients, as well as any product which results,directly or indirectly, from combination, complexation or aggregation ofany two or more of the ingredients, or from dissociation of one or moreof the ingredients, or from other types of reactions or interactions ofone or more of the ingredients. Accordingly, the pharmaceuticalcompositions of the present invention encompass any composition made byadmixing a compound of the present invention and a pharmaceuticallyacceptable excipient (pharmaceutically acceptable carrier).

The term “excipient” refers to a diluent, adjuvant, or vehicle withwhich the therapeutic is administered. Such pharmaceutical excipient canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, including but notlimited to peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a preferred excipient when the pharmaceutical compositionis administered orally. Saline and aqueous dextrose are preferredexcipients when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as liquid excipients for injectablesolutions. Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsions, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andexcipients such as triglycerides. Oral formulation can include standardexcipients such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical excipients are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the therapeutic,preferably in purified form, together with a suitable amount ofexcipient so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Preferably, X³ is O.

Preferably, X is N(R⁴), X¹ is C and X³ is O.

Preferably, X² is C(R⁷R^(7a)).

Preferably, L¹ is selected from the group consisting of

wherein R is H; or C₁₋₄ alkyl; Y is NH; O; or S; and R¹, R^(1a), R²,R^(2a), R³, R^(3a), R⁴, X, X¹, X², have the meaning as indicated above.

Even more preferred, L¹ is selected from the group consisting of

wherein R has the meaning as indicated above.

At least one (up to four) hydrogen is replaced by a group L²-Z. In casemore than one group L²-Z is present each L² and each Z can be selectedindependently. Preferably, only one group L²-Z is present resulting inthe formula D-L¹-L²-Z.

In general, L² can be attached to L¹ at any position apart from thereplacement of the hydrogen marked with an asterisk in formula (I).Preferably, one to four of the hydrogen given by R, R¹ to R⁸ directly oras hydrogen of the C₁₋₄ alkyl or further groups and rings given by thedefinition of R and R¹ to R⁸ are replaced by L²-Z.

Furthermore, L¹ may be optionally further substituted. In general, anysubstituent may be used as far as the cleavage principle is notaffected.

Preferably, one or more further optional substituents are independentlyselected from the group consisting of halogen; CN; COOR⁹; OR⁹; C(O)R⁹;C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a)); S(O)₂R⁹; S(O)R⁹;N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); NO₂; OC(O)R⁹;N(R⁹)C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O)R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of T, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R^(11a)R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein T is optionally substitutedwith one or more R¹⁰, which are the same or different;R¹⁰ is halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²; C(O)N(R¹²R^(12a));S(O)₂N(R¹²R^(12a)); S(O)N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²;N(R¹²)S(O)₂N(R^(12a)R^(12b)); SR₁₂; N(R¹²R^(12a)); NO₂; OC(O)R¹²;N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a); N(R¹²)S(O)R^(12a);N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b)); OC(O)N(R¹²R^(12a)); orC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different;R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or at the end of the carbon chain between the carbon andhydrogen.

L² is a single chemical bond or a spacer. In case L² is a spacer, it ispreferably defined as the one or more optional substituents definedabove, provided that L² is substituted with Z.

Accordingly, when L² is other than a single chemical bond, L²-Z isCOOR⁹; OR⁹; C(O)R⁹; C(O)N(R⁹R^(9a)); S(O)₂N(R⁹R^(9a)); S(O)N(R⁹R^(9a));S(O)₂R⁹; S(O)R⁹; N(R⁹)S(O)₂N(R^(9a)R^(9b)); SR⁹; N(R⁹R^(9a)); OC(O) R⁹;N(R⁹) C(O)R^(9a); N(R⁹)S(O)₂R^(9a); N(R⁹)S(O) R^(9a); N(R⁹)C(O)OR^(9a);N(R⁹)C(O)N(R^(9a)R^(9b)); OC(O)N(R⁹R^(9a)); T; C₁₋₅₀ alkyl; C₂₋₅₀alkenyl; or C₂₋₅₀ alkynyl, wherein T; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—; —O—; —C(O)—; —C(O)N(R¹¹)—;—S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—; —N(R¹¹)S(O)₂N(R^(11a))—;—S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—; —N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—;—N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and —OC(O)N(R¹¹R^(11a));

R⁹, R^(9a), R^(9b) are independently selected from the group consistingof H; Z; T; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; or C₂₋₅₀ alkynyl, wherein T;C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different and wherein C₁₋₅₀alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of T, —C(O)O—;—O—; —C(O)—; —C(O)N(R¹¹)—; —S(O)₂N(R¹¹)—; —S(O)N(R¹¹)—; —S(O)₂—; —S(O)—;—N(R¹¹)S(O)₂N(R^(11a))—; —S—; —N(R¹¹)—; —OC(O)R¹¹; —N(R¹¹)C(O)—;—N(R¹¹)S(O)₂—; —N(R¹¹)S(O)—; —N(R¹¹)C(O)O—; —N(R¹¹)C(O)N(R^(11a))—; and—OC(O)N(R¹¹R^(11a));T is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4 to 7 membered heterocyclyl; or9 to 11 membered heterobicyclyl, wherein t is optionally substitutedwith one or more R¹⁰, which are the same or different;R¹⁰ is Z; halogen; CN; oxo (═O); COOR¹²; OR¹²; C(O)R¹²;C(O)N(R¹²R^(12a)); N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²; S(O)₂N(R¹²R^(12a));S(O)N(R¹²R^(12a)); S(O)₂R¹²; S(O)R¹²; N(R¹²)S(O)₂N(R^(12a)R¹²); SR¹²;N(R¹²R^(12a)); NO₂; OC(O)R¹²; N(R¹²)C(O)R^(12a); N(R¹²)S(O)₂R^(12a);N(R¹²)S(O)R^(12a); N(R¹²)C(O)OR^(12a); N(R¹²)C(O)N(R^(12a)R^(12b));OC(O)N(R¹²R^(12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different;R¹¹, R^(11a), R¹², R^(12a), R^(12b) are independently selected from thegroup consisting of H; Z; or C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent;provided that one of R⁹, R^(9a), R^(9b), R¹⁰, R¹¹, R^(11a), R¹²,R^(12a), R^(12b), is Z.

More preferably, L² is a C₁₋₂₀ alkyl chain, which is optionallyinterrupted by one or more groups independently selected from —O—; andC(O)N(R^(3aa)); optionally substituted with one or more groupsindependently selected from OH; and C(O)N(R^(3aa)R^(3aaa)); and whereinR^(3aa), R^(3aaa) are independently selected from the group consistingof H; and C₁₋₄ alkyl.

Preferably, L² has a molecular weight in the range of from 14 g/mol to750 g/mol.

Preferably, L² is attached to Z via a terminal group selected from

In case L² has such terminal group it is furthermore preferred that L²has a molecular weight in the range of from 14 g/mol to 500 g/molcalculated without such terminal group.

Preferably, L is represented by formula (Ia)

wherein R⁴, L², and Z have the meaning as indicated above, and whereinR^(3aa), R^(3aaa) independently selected from the group consisting of H;and C₁₋₄ alkyl; or are joined together with the nitrogen atom to whichthey are attached to form a 4 to 7 membered heterocycle. Preferably, R⁴is H; or methyl.

Preferably, L is represented by formula (Ib)

wherein R¹, R^(1a), R⁴, L² and Z have the meaning as indicated above,and wherein R^(3aa) is H; or C₁₋₄ alkyl. Preferably, R⁴ is H; or methyl.

Preferably, R¹ in formula (I) is L²-Z.

Preferably, R³ in formula (I) is L²-Z.

Preferably, R³, R^(3a) in formula (I) are joined together with thenitrogen atom to which they are attached to form a 4 to 7 memberedheterocycle, wherein the heterocycle is substituted with L²-Z.

Preferably, D-H is a small molecule bioactive agent or a biopolymer.

Preferably, D-H is a biopolymer selected from the group of biopolymersconsisting of proteins, polypeptides, oligonucleotides, and peptidenucleic acids.

“Oligonucleotides” means either DNA, RNA, single-stranded ordouble-stranded, siRNA, miRNA, aptamers, and any chemical modificationsthereof with preferably 2 to 1000 nucleotides. Modifications include,but are not limited to, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid ligandbases or to the nucleic acid ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,methylations, unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine and the like. Modifications can alsoinclude 3′ and 5′ modifications such as capping and change ofstereochemistry.

Preferably, D-H is a polypeptide selected from the group of polypeptidesconsisting of ACTH, adenosine deaminase, agalsidase, alfa-1 antitrypsin(AAT), alfa-1 proteinase inhibitor (API), alteplase, amylins (amylin,symlin), anistreplase, ancrod serine protease, antibodies (monoclonal orpolyclonal, and fragments or fusions), antithrombin III, antitrypsins,aprotinin, asparaginases, atosiban, biphalin, bivalirudin,bone-morphogenic proteins, bovine pancreatic trypsin inhibitor (BPTI),cadherin fragments, calcitonin (salmon), collagenase, complement C1esterase inhibitor, conotoxins, cytokine receptor fragments, DNase,dynorphine A, endorphins, enfuvirtide, enkephalins, erythropoietins,exendins, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX,fibrinolysin, fibroblast growth factor (FGF), growth hormone releasingpeptide 2 (GHRP2), fusion proteins, follicle-stimulating hormones,gramicidin, ghrelin, desacyl-ghrelin, granulocyte colony stimulatingfactor (G-CSF), galactosidase, glucagon, glucagon-like peptides,glucocerebrosidase, granulocyte macrophage colony stimulating factor(GM-CSF), human heat shock proteins (HSP), phospholipase-activatingprotein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis Bvaccines, hirudin, human serine protease inhibitor, hyaluronidases,idurnonidase, immune globulins, influenza vaccines, interleukins (1alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12, 13, 21), IL-1 receptor antagonist(rhIL-1ra), insulins, insulin like growth factors, insulin-like growthfactor binding protein (rhIGFBP), interferons (alfa 2a, alfa 2b, alfa2c, beta 1a, beta 1b, gamma 1a, gamma 1b), intracellular adhesionmolecule, keratinocyte growth factor (KGF), P-selectin glycoproteinligand (PSGL), transforming growth factors, lactase, leptin, leuprolide,levothyroxine, luteinizing hormone, lyme vaccine, natriuretic peptides(ANP, BNP, CNP and fragments), neuropeptide Y, pancrelipase, pancreaticpolypeptide, papain, parathyroid hormone, PDGF, pepsin, peptide YY,platelet activating factor acetylhydrolase (PAF-AH), prolactin, proteinC, thymalfasin, octreotide, secretin, sermorelin, soluble tumor necorsisfactor receptor (TNFR), superoxide dismutase (SOD), somatropins (growthhormone), somatoprim, somatostatin, streptokinase, sucrase,terlipressin, tetanus toxin fragment, tilactase, thrombins, thymosin,thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF),TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH,urodilatin, urate oxidase, urokinase, vaccines, vascular endothelialgrowth factor (VEGF), vasoactive intestinal peptide, vasopressin,ziconotide, lectin and ricin.

Preferably, D-H is a protein prepared by recombinant DNA technologies.

Preferably, D-H is a protein selected from the group of proteinsconsisting of antibody fragments, single chain antigen binding proteins,catalytic antibodies and fusion proteins.

Preferably, D-H is a small molecule bioactive agent selected from thegroup of agents consisting of central nervous system-active agents,anti-infective, anti-allergic, immunomodulating, anti-obesity,anticoagulants, antidiabetic, anti-neoplastic, antibacterial,anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal,vasodilating, vasoconstricting, and cardiovascular agents with at leastone primary or secondary amino group.

Preferably, D-H is a small molecule bioactive agent selected from thegroup of agents consisting of acarbose, alaproclate, alendronate,amantadine, amikacin, amineptine, aminoglutethimide, amisulpride,amlodipine, amotosalen, amoxapine, amoxicillin, amphetamine,amphotericin B, ampicillin, amprenavir, amrinone, anileridine,apraclonidine, apramycin, articaine, atenolol, atomoxetine, avizafone,baclofen, benazepril, benserazide, benzocaine, betaxolol, bleomycin,bromfenac, brofaromine, carvedilol, cathine, cathinone, carbutamid,cefalexine, clinafloxacin, ciprofloxacin, deferoxamine, delavirdine,desipramine, daunorubicin, dexmethylphenidate, dexmethylphenidate,diaphenylsulfon, dizocilpine, dopamin, dobutamin, dorzolamide,doxorubicin, duloxetine, eflornithine, enalapril, epinephrine,epirubicin, ergoline, ertapenem, esmolol, enoxacin, ethambutol,fenfluramine, fenoldopam, fenoterol, fingolimod, flecainide,fluvoxamine, fosamprenavir, frovatriptan, furosemide, fluoexetine,gabapentin, gatifloxacin, gemiflocacin, gentamicin, grepafloxacin,hexylcaine, hydralazine, hydrochlorothiazide, icofungipen, idarubicin,imiquimod, inversine, isoproterenol, isradipine, kanamycin A, ketamin,labetalol, lamivudine, levobunolol, levodopa, levothyroxine, lisinopril,lomefloxacin, loracarbef, maprotiline, mefloquine, melphalan, memantine,meropenem, mesalazine, mescaline, methyldopa,methylenedioxymethamphetamine, metoprolol, milnacipran, mitoxantron,moxifloxacin, norepinephrine, norfloxacin, nortriptyline, neomycin B,nystatin, oseltamivir, pamidronic acid, paroxetine, pazufloxacin,pemetrexed, perindopril, phenmetrazine, phenelzine, pregabalin,procaine, pseudoephedrine, protriptyline, reboxetine, ritodrine,sabanibicin, salbutamol, serotonin, sertraline, sitagliptin, sotalol,spectinomycin, sulfadiazin, sulfamerazin, sertraline, sprectinomycin,sulfalen, sulfamethoxazol, tacrine, tamsulosin, terbutaline, timolol,tirofiban, tobramycin, tocainide, tosufloxacin, trandolapril, tranexamicacid, tranylcypromine, trimerexate, trovafloxacin, valaciclovir,valganciclovir, vancomycin, viomycin, viloxazine, and zalcitabine.

Preferably, Z is a polymer of at least 500 Da or a C₈₋₁₈ alkyl group.

Preferably, Z is selected from the group of optionally crosslinkedpolymers consisting of poly(propylene glycol), poly(ethylene glycol),dextran, chitosan, hyaluronic acid, alginate, xylan, mannan,carrageenan, agarose, cellulose, starch, hydroxyalkyl starch (HAS),poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), poly(orthoesters), poly(carbonates), poly(urethanes), poly(acrylic acids),poly(acrylamides), poly(acrylates), poly(methacrylates),poly(organophosphazenes), polyoxazoline, poly(siloxanes), poly(amides),poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters),poly(iminocarbonates), poly(amino acids), collagen, gelatin, hydrogel ora blood plasma protein, and copolymers thereof.

Preferably, Z is a protein.

Preferably, Z is a protein selected from the group consisting ofalbumin, transferrin, immunoglobulin.

Preferably, Z is a linear or branched poly(ethylene glycol) with amolecular weight from 2,000 Da to 150,000 Da.

Even more preferred is a prodrug of the present invention, wherein D-His a GLP-1 receptor agonist; L is L¹ represented by formula (I) asindicated above; and Z is a hydrogel. Even more preferably, in formula(I) X is N(R⁴), X¹ is C and X³ is O. Even more preferably, L isrepresented by formula (Ia) as indicated above.

GLP-1 is one of the intestinal peptide hormones that are released intothe circulatory system after food intake. It augments the postprandialrelease of insulin, when nutrition (especially carbohydrates) areabsorbed and their level postprandially elevated. GLP-1 associates withGLP-1 receptor sites located on pancreatic β-cells and elevatesendogenous cAMP levels in a dose dependent manner. In isolated ratislets in the presence of above normoglycemic glucose levels, GLP-1stimulates the release of insulin. A therapeutic potential for GLP-1 intype 2 diabetes patients was suggested before, owing to the profoundefficacy of this insulinotropic peptide to stimulate secretion ofinsulin when glucose levels are elevated and to cease doing so uponreturn to normoglycemia. The antidiabetogenic effect of glucagon-likepeptide-1 (7-36) amide in normal subjects and patients with diabetesmellitus is described e. g. in N. Engl. J. Med. 326(20):1316-1322. Invitro studies and animal experiments suggest that GLP-1 improves insulinsensitivity and has an anabolic effect on pancreatic β-cells. In humans,GLP-1 was also reported to suppress glucagon secretion, decelerategastric emptying, and induce satiety, leading to weight loss ifadministered for weeks and months.

Exendin-4 is reported to associate with GLP-1 receptors located onpancreatic beta-cells with 2.5 times higher affinity than GLP-1. Inisolated rat islets and beta-cells in presence of glucose, exendinenhances secretion of insulin in a dose-dependent fashion. Exendin-4 isa high potency agonist and truncated exendin-(9-39)-amide an antagonistat the glucagon-like peptide 1-(7-36)-amide receptor ofinsulin-secreting beta-cells (see J. Biol. Chem. 268(26):19650-19655).Studies in type 2 diabetic rodents revealed that exendin-4 is 5530-foldmore potent than GLP-1 in lowering blood glucose levels. Also, theduration of glucose-lowering action after a single administration ofexendin-4 is significantly longer compared to GLP-1 (see e.g. Diabetes48(5):1026-1034). Plasma half-life of exendin-4 in humans was describedto be only 26 minutes. Exendin-4 reduces fasting and postprandialglucose and decreases energy intake in healthy volunteers (see e.g. Am.J. Physiol. Endocrinol. Metab. 281(1): E155-61).

Accordingly in an even more preferred embodiment the GLP-1 receptoragonist is Exendin-4.

Hydrogels to be used are known in the art. Suitable hydrogels may beused which are described in WO-A 2006/003014. Accordingly, a hydrogelmay be defined as a three-dimensional, hydrophilic or amphiphilicpolymeric network capable of taking up large quantities of water. Thenetworks are composed of homopolymers or copolymers, are insoluble dueto the presence of covalent chemical or physical (ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork structure and physical integrity. Hydrogels exhibit athermodynamic compatibility with water which allow them to swell inaqueous media. The chains of the network are connected in such a fashionthat pores exist and that a substantial fraction of these pores are ofdimensions between 1 nm and 1000 nm.

Another object of the present invention is a pharmaceutical compositioncomprising a prodrug of the present invention or a pharmaceutical saltthereof together with a pharmaceutically acceptable excipient.

Yet another object of the present invention is a prodrug of the presentinvention or a pharmaceutical composition of the present invention foruse as a medicament.

Yet another object of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient in need ofthe treatment of one or more conditions comprising administering to saidpatient a therapeutically effective amount of a prodrug of the presentinvention or a pharmaceutical composition of the present invention or apharmaceutically acceptable salt thereof.

Another object of the present invention is a prodrug precursor offormula Act-L, wherein L has the meaning as indicated above and Act is aleaving group.

Preferably, Act is chloride, bromide, fluoride, nitrophenoxy,imidazolyl, N-hydroxysuccinimidyl, N-hydroxybenzotriazolyl,N-hydroxyazobenzotriazolyl, pentafluorophenoxy, 2-thiooxo-thiazolidinyl,or N-hydroxysulfosuccinimidyl.

EXAMPLES Materials and Methods

Materials: Side chain protected Exendin-4 (J. Eng et al., J. Biol. Chem.1992, 267, 11, 7402-7405) on Rink amide resin, side chain protectedBNP-32a (human, Cys10 and Cys26 exchanged for Ala) on chlorotritylresin, side chain protected BNP-32b with ivDde side-chain protectinggroup on Lys14 (human, Cys10 and Cys26 exchanged for Ala) onchlorotrityl resin, and side chain protected human growth hormonereleasing factor fragment 1-29 amide (GRF(1-29)) on Rink amide (eachsynthesized by Fmoc-strategy) were obtained from Peptide SpecialtyLaboratories GmbH, Heidelberg, Germany. Standard side chain protectinggroups were used except for Lys27 of Exendin-4 and Lys21 of GRF(1-29)where Mint side-chain protecting groups were used.

40 kDa methoxy poly(ethylene glycol) maleimido-propionamide (PEG40kDa-maleimide) was obtained from Chirotech Technology Ltd, Cambridge,UK.

2-Chlorotrityl chloride resin, Sieber amide resin and amino acids werefrom Merck Biosciences GmbH, Schwalbach/Ts, Germany, if not statedotherwise. Fmoc-D-Homocysteine(Trt)-OH and S-Trityl-3-mercaptopropionicacid (Trt-MPA) were obtained from Bachem AG, Bubendorf, Switzerland.O—(N-Fmoc-2-aminoethyl)-O′-(2-carboxyethyl)-undecaethyleneglycol(Fmoc-Pop-OH) was obtained from Polypure AS, Oslo, Norway.Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine (Fmoc-Acp-OH) waspurchased from NeoMPS SA, Strasbourg, France.cis-Cyclohexane-1,2-dicarboxylic anhydride was obtained from Alfa AesarGmbH & Co KG, Karlsruhe, Germany.

All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen,Germany.

Solid phase synthesis was performed on 2-Chlorotrityl chloride resinwith a loading of 1.3 mmol/g or Sieber amide resin with a loading of0.55 mmol/g. Syringes equipped with polypropylene frits were used asreaction vessels.

Loading of the first amino acid to resins was performed according tomanufacturer's instructions.

Fmoc Deprotection:

For Fmoc protecting-group removal, the resin was agitated with 2/2/96(v/v/v) piperidine/DBU/DMF (two times, 10 min each) and washed with DMF(ten times).

ivDde Deprotection:

For ivDde protecting-group removal, the resin was agitated with 98/2(v/v) DMF/hydrazine hydrate (3 times, 10 min each) and washed with DMF(ten times).

Boc Protection:

The N-terminus of a peptide was boc-protected by agitating the resinwith 30 eq (boc)₂O and 60 eq pyridine in DCM. After 1 h the resin waswashed with DCM (10 times).

Standard Coupling Condition for Acids:

Coupling of acids (aliphatic acids, Fmoc-amino acids) to free aminogroups on resin was achieved by agitating resin with 3 eq of acid, 3 eqPyBOP and 6 eq DIEA in relation to free amino groups on resin(calculated based on theoretical loading of the resin) in DMF at roomtemperature. After 1 hour resin was washed with DMF (10 times).

3-Maleimido Propionic Acid Coupling:

Coupling of 3-maleimido propionic acid to free amino groups on resin wasachieved by agitating resin with 2 eq of acid, 2 eq DIC and 2 eq HOBt inrelation to free amino groups in DMF at room temperature. After 30 min,resin was washed with DMF (10 times).

Standard Protocol for Synthesis of Ureas on Resin:

Synthesis of ureas on resin was achieved by agitating resin with 2.5 eqof bis(pentafluorophenyl) carbonate, 5 eq DIEA, and 0.25 eq DMAP inrelation to free amino groups in DCM/ACN 1/1 at room temperature. After15 min resin was washed with DMF (10 times). 5 eq of amine was dissolvedin DMF. Mixture was added to resin and agitated for 60 min at roomtemperature. Resin was washed with DMF (10 times).

Cleavage Protocol for Sieber Amide Resin:

Upon completed synthesis, the resin was washed with DCM (10 times),dried in vacuo and treated repeatedly (five times a 15 minutes) with97/2/1 (v/v) DCM/TES/TFA. Eluates were combined, volatiles were removedunder a nitrogen stream and product was purified by RP-HPLC. HPLCfractions containing product were combined and lyophilized.

Cleavage Protocol for 2-Chlorotrityl Chloride Resin:

Upon completed synthesis, the resin was washed with DCM, dried in vacuoand treated two times for 30 minutes with 6/4 (v/v) DCM/HFIP. Eluateswere combined, volatiles were removed under a nitrogen stream andproduct was purified by RP-HPLC. HPLC fractions containing product werecombined and lyophilized.

Cleavage Protocol for Rink Amide Resin:

Upon completed synthesis, the resin was washed with DCM, dried in vacuoand treated with 2 ml of TFA cleavage cocktail (TFA/TES/Water/DTT95/2/2/1) per 100 mg resin for 60 min at room temperature. Volatileswere removed under a nitrogen stream. Unpolar side products andprotecting groups were removed by precipitating peptide from diethylether. Precipitate was dried in vacuo and dissolved in ACN/water 1/1 andpurified by RP-HPLC.

Amine containing products obtained as TFA salts were converted to thecorresponding HCl salts using ion exchange resin (Discovery DSC-SAX,Supelco, USA). This step was performed in case the residual TFA wasexpected to interfere with e.g. a subsequent coupling reactions.

RP-HPLC Purification:

RP-HPLC was done on a 100×20 or a 100×40 mm C18 ReproSil-Pur 300 ODS-35μ column (Dr. Maisch, Ammerbuch, Germany) connected to a Waters 600HPLC System and Waters 2487 Absorbance detector. Linear gradients ofsolution A (0.1% TFA in H₂O) and solution B (0.1% TFA in acetonitrile)were used. HPLC fractions containing product were lyophilized.

Analytics: Electrospray ionization mass spectrometry (ESI-MS) wasperformed on a Waters ZQ 4000 ESI instrument and spectra were, ifnecessary, interpreted by Waters software MaxEnt.

Size exclusion chromatography (SEC) was performed using an AmershamBioscience AEKTAbasic system equipped with a Superdex200 10/300 column(Amersham Bioscience/GE Healthcare), if not stated otherwise. 10 mMsodium phosphate, 140 mM NaCl, pH 7.4, 3 mM EDTA was used as mobilephase

For Cation Exchange Chromatography, an Amersham Bioscience AEKTAbasicsystem was equipped with a Source 15S filled HR16/10 column (AmershamBioscience/GE Healthcare).

Desalting was performed using an Amersham Bioscience AEKTAbasic systemequipped with a HiPrep 26/10 Desalting column and 0.1% acetic acid inwater as mobile phase.

In vitro linker hydrolysis and release of drug: Compounds were dissolvedin buffer A (10 mM sodium phosphate, 140 mM NaCl, pH 7.4, 3 mM EDTA) orbuffer B (0.1 M Acetat 3 mM EDTA, pH 4.0), and solution was filteredthrough a 0.2 μm filter and incubated at 37° C. Samples were taken attime intervals and analyzed by RP-HPLC at 215 nm and ESI-MS. UV-signalscorrelating to liberated drug molecule were integrated and plottedagainst incubation time. In case of identical retention times of prodrugand drug, ratio of mass signals was used to determine release kinetics.

For hydrogel conjugates, compounds were suspended in buffer A andincubated at 37° C. Samples were taken after centrifugation of thesuspension and analyzed by RP-HPLC at 215 nm. UV-signals correlating toliberated drug molecule were integrated and plotted against incubationtime.

Curve-fitting software was applied to estimate the correspondinghalftime of release.

Example 1 Synthesis of Fatty Acid Carrier (1)

1 was synthesized on sieber amide resin (477 mg, 0.262 mmol) by couplingof Fmoc-Lys(ivDde)-OH, fmoc deprotection, coupling of dodecanoic acid,ivDde deprotection, coupling of Fmoc-Pop-OH, fine deprotection, couplingof 3-maleimido propionic acid, cleavage from resin and purification asdepicted above and described in “Materials and Methods”.

Yield: 128 mg (0.119 mmol).

MS: m/z 1101.0=[M+Na]⁺ (MW calculated=1078.4 g/mol).

Example 2 Synthesis of Linker Reagent (2)

Linker reagent 2 was synthesized on 3-chlorotrityl chloride resin (300mg, 0.39 mmol) by loading of resin with Fmoc-Cys(Trt)-OH, fmocdeprotection, and on-resin urea formation usingN,N-dimethyl-ethylenediamine as amine, cleavage from resin as depictedabove and described in “Materials and Methods”. For RP-HPLC separation,0.01% HCl in water was used as solution A and 0.01% HCl in acetonitrilewas used as solution B.

Yield: 82 mg of HCl salt (0.16 mmol).

MS: m/z 478.2=[M+H]⁺ (MW calculated=477.6 g/mol).

Example 3 Synthesis of Exendin-4 Linker Intermediate (3)

2 (14 mg, 0.027 mmol), PyBOP (14 mg, 0.027 mmol), and DIEA (17 μl, 0.10mmol) were dissolved in 0.2 ml of dry DMF. Mixture was added to 22 mgside-chain protected Exendin-4 on-resin (0.1 mmol/g, 2.2 μmol) andagitated for 30 min at room temperature. Resin was washed with DMF (10times) and DCM (10 times). 3 was cleaved from resin and purified byRP-HPLC as described in “Materials and Methods”.

Yield: 1.7 mg 3 as TFA salt (0.38 μmol).

MS: m/z 1468.7=[M+3H]³⁺ (MW calculated=4403 g/mol).

Example 4 Synthesis of Fatty Acid-PEG-Linker-Exendin-4 Conjugate (4)

3 (1.7 mg, 0.38 μmol) and 1 (0.6 mg, 0.58 μmol) were dissolved in 500 μlof acetonitrile/water 7/3 (v/v). 40 μl of 0.5 M phosphate buffer (pH7.4) were added and the mixture was incubated at RT for 10 min.Conjugate 4 was purified by RP-HPLC.

MS: m/z 1828.7=[M+3H]³⁺ (MW calculated=5480 g/mol).

Example 5 Synthesis of Linker Intermediate (5a)

Fmoc-Acp-OH.2 HCl (100 mg, 0.21 mmol) was suspended in 400 μl DMF/DMSO1/1 (v/v). S-tritylcysteamine.HCl (75 mg, 0.21 mmol), PyBOP (109 mg,0.21 mmol) and DIEA (146 μl, 0.86 mmol) were added and mixture wasagitated for 60 min at RT. Fmoc group was removed by adding 75 μlpiperidine and 25 μl DBU. After 15 min mixture was hydrolyzed andacidified (AcOH) and compound was purified by RP-HPLC. Afterlyophilization 98 mg (0.14 mmol, double TFA salt) were obtained.

MS: m/z 511.6=[M+Na]⁺ (MW calculated=488.7 g/mol).

Synthesis of Cis-Cyclohexane Diacarboxylic Acid Amoxapine Monoamide (5b)

Amoxapine (200 mg, 0.64 mmol) and cis-cyclohexane-1,2-dicarboxylicanhydride (108 mg, 0.70 mmol) were dissolved in 700 μl of dry DMF.Pyridine (130 μl, 1.6 mmol) was added and mixture was stirred for 60 minat RT. Mixture was quenched with 2 ml of acetonitrile/acetic acid/water(1/1/1) and purified by RP-HPLC. After lyophilization 344 mg (0.49 mmol,double TFA salt) of 5b were obtained.

MS: m/z 468.5=[M+H]⁺ (MW calculated=468.0 g/mol).

Synthesis of Linker-Amoxapine Conjugate (5c)

5b (7 mg, 0.010 mmol) was preactivated by incubating with PyBOP (12.5mg, 0.024 mmol) and DIEA (5 μl, 0.03 mmol) in 200 μl of dry DMF for 45min at RT. 5a (20 mg, 0.028 mmol) and DIEA (15 μl, 0.09 mmol) were addedand mixture was incubated for further 60 min. Mixture was quenched with0.5 ml of acetonitrile/acetic acid/water (1/1/1) and purified byRP-HPLC. After lyophilization 3 mg (0.0026 mmol, double TFA salt) of 5cwere obtained.

MS: m/z 939.3=[M+H]⁺ (MW calculated=938.6 g/mol).

For trityl deprotection, lyophilisate was incubated in 1 ml HFIP and 3μl TES for 30 min. Mixture was evaporated and thiol was purified byRP-HPLC. After lyophilization 2 mg (2.2 μmol, double TFA salt) ofamoxapine-linker conjugate 5c were obtained.

MS: m/z 697.1=[M+H]⁺ (MW calculated=696.3 g/mol).

Synthesis of Fatty Acid-PEG-Amoxapine Conjugate (5)

Amoxapine-linker conjugate 5c (2 mg, 2.2 μmol) and 1 (3.5 mg, 3.2 μmol)were dissolved in 900 μl of acetonitrile/water 7/3 (v/v). 60 μl of 0.5 Mphosphate buffer (pH 7.4) were added and the mixture was incubated at RTfor 10 min. 5 was purified by RP-HPLC.

MS: m/z 1774.9=[M+H]⁺ (MW calculated=1774.7 g/mol).

Example 6 Synthesis of linker reagent (6)

Fmoc-Asp(tBu)-OH (411 mg, 1 mmol), HOBt (153 mg, 1 mmol), and DIC (160μl, 1 mmol) were dissolved in 2 ml of DMF and incubated for 10 min atRT. N,N-dimethyl ethylenediamine (160 μl, 1.5 mmol) was added andstirred at RT for 30 min. Acetic acid (300 μl) was added andFmoc-Asp(tBu)-NH—(CH₂)₂—N(CH₃)₂ was purified by RP-HPLC.

Yield: 220 mg (0.46 mmol)

MS Fmoc-Asp(tBu)-NH—(CH₂)₂—N(CH₃)₂: m/z 504.6=[M+Na]⁺ (MWcalculated=481.6 g/mol).

Fmoc-Asp(tBu)-NH—(CH₂)₂—N(CH₃)₂ (220 mg, 0.46 mmol) was dissolved in 3ml of 98/2 (v/v) TFA/TES. After 30 min the solvent was removed under anitrogen stream and 6 was purified by RP-HPLC using 0.01% HCl in wateras solvent A and 0.01% HCl in acetonitril as solvent B.

Yield: 146 mg (0.32 mmol, HCl salt)

MS: m/z 426.5=[M+H]⁺ (MW calculated=425.5 g/mol).

Example 7 Synthesis of Linker Reagents 7a and 7b

Synthesis of 7a:

Fmoc-Asp(tBu)-OH (300 mg, 0.73 mmol), HOBt (1112 mg, 0.73 mmol), and DIC(117 μl, 0.73 mmol) were dissolved in 2 ml of DMF and incubated for 10min at RT. Boc-ethylenediamine (230 mg, 1.44 mmol) was added and stirredat RT for 30 min. Acetic acid (300 μl) was added andFmoc-Asp(tBu)-NH—(CH₂)₂—NH-boc was purified by RP-HPLC.

Yield: 205 mg (0.37 mmol)

MS intermediate: m/z 576.6=[M+Na]⁺ (MW calculated=553.7 g/mol).

Fmoc-Asp(tBu)-NH—(CH₂)₂—NH-boc (205 mg, 0.37 mmol) was dissolved in 3 mlof 98/2 (v/v) TFA/TES. After 30 min the solvent was removed under anitrogen stream and Fmoc-Asp(H)—NH—(CH₂)₂—NH₂ was purified by RP-HPLC.

Yield: 140 mg (0.27 mmol, TFA salt)

MS intermediate: m/z 398.8=[M+H]⁺ (MW calculated=397.4 g/mol).

Fmoc-Asp(H)—NH—(CH₂)₂—NH₂ (140 mg, 0.27 mmol, TFA salt) was dissolved in1 ml of DMF and DIEA (140 μl, 0.81 mmol) and boc₂O (100 mg, 0.46 mmol)added. The solution was stirred at RT for 15 min and then acidified withacetic acid (300 μl). 7a was purified by RP-HPLC.

Yield 7a: 120 mg (0.24 mmol)

MS 7a: m/z 520.5=[M+Na]⁺ (MW calculated=497.6 g/mol).

7b was synthesized as described above except for the use ofH₂N—(CH₂)₂—N(CH₃)-boc instead of boc-ethylenediamine as amine in thefirst step.

Yield 7b: 115 mg

MS 7b: m/z 534.5=[M+Na]⁺ (MW calculated=511.6 g/mol).

Example 8 Synthesis of Exendin-Linker Conjugates 8a, 8b and 8c

Synthesis of 8a:

7a (30 mg, 60 μmol), HOBt (9 mg, 60 μmol), DIEA (12 μl, 70 μmol), andDIC (10 μl, 63 μmol) were dissolved in 200 μl of DMF and immediatelyadded to side-chain protected Exendin-4 on resin (40 mg, 4 μmol) andincubated for 1 h at room temperature. Resin was washed ten times withDMF and then incubated for 5 min with 500 μl of 1/1/2 aceticanhydride/pyridine/DMF. Resin was washed 10 times with DMF and fmocgroup was removed. Trt-mercaptopropionic acid was coupled and 8a wascleaved from resin and purified by RP-HPLC.

Yield: 3.6 mg

MS 8a: m/z 1108.5=[M+4H]⁴⁺; 1477.8=[M+3H]³⁺ (MW calculated=4432 g/mol).

8b was synthesized as described above for 8a except for the use of 7binstead of 7a.

Yield: 3.5 mg

MS 8b: m/z 1112.5=[M+4H]⁴⁺; 1482.5=[M+3H]³⁺ (MW calculated=4446 g/mol).

8c was synthesized as described above for 8a except for the use of 6instead of 7a.

Yield: 3.2 mg

MS 8c: m/z 1116.2=[M+4H]⁴⁺; 1487.8=[M+3H]³⁺ (MW calculated=4460 g/mol).

Example 9 Synthesis of PEG40 kDa-linker-Exendin conjugates 9a, 9b, and9c

Synthesis of 9a:

8a (3.6 mg) was dissolved in 300 μl 2/1 water/acetonitrile and 50 mgPEG40 kDa-maleimide was added. 100 μl 0.25 M sodium phosphate buffer pH7 were added and after 5 min the solution was acidified with 50 μlacetic acid.

9a was purified by ion exchange chromatography using 10 mM sodiumcitrate pH 3 as solvent A and 10 mM sodium citrate pH 3 and 1 M NaCl assolvent B and a step-gradient (0 to 40% B). Fractions containing 9a weredesalted and lyophilized:

Yield: 14 mg

9b was synthesized as described above except for the use of 8b.

Yield: 15 mg

9c was synthesized as described above except for the use of 8c.

Yield: 13 mg

Example 10 Synthesis of Fatty Acid-Linker-Exendin Conjugate 10

8c (1 mg) was dissolved in 100 μl 1/1 acetonitrile/water and 1 (1 mg) in100 μl of 3/1 acetonitrile/water was added. 100 μl of 0.25 M sodiumphosphate buffer was added, the reaction was stirred for 5 min, afterwhich 10 was purified by RP-HPLC.

Yield: 1.3 mg

MS 10: m/z 1385.9=[M+4H]⁴⁺; 1846.3=[M+3H]³⁺ (MW calculated=5528.3g/mol).

Example 11 Synthesis of NHS-Activated Linker Reagent 11

7b (20 mg, 40 μmol), N,N′-dicyclohexylcarbodiimide (10 mg, 48 μmol), andNHS (8 mg, 70 μmol) were dissolved in 300 μl of dry DCM and stirred atRT for 1 h. Solvent was removed under a nitrogen stream and 11 waspurified by RP-HPLC and lyophilized.

Yield: 22 mg (36 μmol)

MS: m/z 631.5=[M+Na]⁺ (MW calculated=608.7 g/mol).

Example 12 Synthesis of Linker-Exendin(Fluorescein) Conjugate (12a) andLinker-GRF(1-29)(Fluorescein) Conjugate (12b)

6 (60 mg, 130 μmol HCl salt), HOBt (20 mg, 130 μmol), DIEA (40 μl, 230μmol), and DIC (20 μl, 126 μmol) were dissolved in 700 μl of DMF andimmediately added to side-chain protected Exendin-4 on resin (120 mg, 12μmol) and incubated for 1 h at room temperature. Resin was washed tentimes with DMF and then incubated for 5 min with 1 ml of 1/1/2 (v/v/v)acetic anhydride/pyridine/DMF. Resin was washed ten times with DMF andfmoc group was removed. Trt-mercaptopropionic acid was coupled accordingto standard coupling method and resin was washed five times with DMF andten times with DCM. Mmt protecting group of Lys27 was removed byincubation of resin five times in 2 ml of 9/1 (v/v) DCM/HFIP for 5 min.Resin was washed five times with DCM and five times with DMF and5,6-carboxy-flourescein-NHS ester (20 mg, 42 μmol) and DIEA (20 μl, 115μl) in 300 μl DMF were added to resin and incubated for 30 min. 12a wascleaved from resin and purified by RP-HPLC

Yield: 12 mg

MS 12a: m/z 1205.9=[M+4H]⁴⁺; 1607.0=[M+3H]³⁺ (MW calculated=4818.3g/mol). 12b was synthesized as described for 12a except for the use ofGRF(1-29) on resin (120 mg, 12 μmol).

Yield: 11 mg

MS 12b: m/z 998.6=[M+4H]⁴⁺; 1330.5=[M+3H]³⁺ (MW calculated=3989.6g/mol).

Synthesis of Mercaptopropionyl-Exendin(Fluorescein) (12c) andMercaptopropionyl-GRF(1-29)(Fluorescein) (12d)

Trt-mercaptopropionic acid was coupled according to standard couplingmethod to side-chain protected Exendin-4 on resin (120 mg, 12 μmol). Mmtprotecting group removal of Lys27 and 5,6-carboxy-flourescein-NHS estercoupling was performed as described for 12a. 12c was cleaved from resinand purified by RP-HPLC

Yield: 13 mg

MS 12c: m/z 1545.6=[M+3H]³ (MW calculated=4633 g/mol).

12d was synthesized as described for 12c except for the use of GRF(1-29)on resin (120 mg, 12 μmol).

Yield: 11 mg

MS 12d: m/z 1269.1=[M+3H]³⁺ (MW calculated=3804.3 g/mol).

Example 13 Synthesis of Reversible PEG40 kDa-Linker-Exendin(Fluorescein)Conjugate (13a) and Reversible PEG40 kDa-Linker-GRF(1-29)(Fluorescein)Conjugate (13b)

12a (12 mg) was dissolved in 500 μl of 1/1 acetonitrile/water and 120 mgPEG40 kDa-maleimide in 1 ml of 1/1 acetonitrile/water was added. 300 μlof 0.25 M sodium phosphate buffer pH 7.0 were added and solution wasacidified after 10 min with 300 μl acetic acid. 13a was purified bycation exchange chromatography, desalted, and then lyophilized.

Yield: 51 mg

13b was synthesized as described for 13a except for the use of 12binstead of 12a.

Yield: 46 mg

Synthesis of Permanent PEG40 kDa-Exendin(Fluorescein) Conjugate (13c)and Permanent PEG40 kDa-GRF(1-29)(Fluorescein) Conjugate (13d)

13c was synthesized as described for 13a except for the use of 12cinstead of 12a.

Yield: 55 mg

13d was synthesized as described for 13a except for the use of 12dinstead of 12a.

Yield: 45 mg

Example 14 Synthesis of Linker-GRF(1-29) Conjugate 14

14 was synthesized as described for 8c except for the use of side-chainprotected GRF(1-29) resin.

Yield: 10 mg

MS 14: m/z 908.2=[M+4H]⁴⁺; 1211.2=[M+3H]³⁺ (MW calculated=3631.3 g/mol).

Example 15 Synthesis of PEG40 kDa-Linker-GRF(1-29) Conjugate (15)

15 was synthesized as described for 9c except for the use of 14 and 10mM sodium citrate pH 4 as solvent A and 10 mM sodium citrate pH 4 and 1M sodium chloride as solvent B for cation exchange chromatography.

Yield: 11 mg

Example 16 Synthesis of Linker Intermediate 16

N,N-dimethylethylenediamine (198 μL, 1.8 mmol) and NaCNBH₃ (58 mg, 0.9mmol) were dissolved in methanol (5 mL) and brought to pH 5.5 byaddition of AcOH (250 μL). A suspension of 2,4,6,-trimethoxybenzaldehyde(294 mg, 1.5 mmol) in EtOH (5 mL) was added and the reaction was stirredat RT for 1 h. 5 N HCl (0.5 mL) was added and the mixture was stirredfor further 12 h. The solvent was removed under reduced pressure; theresidue was dissolved in sat. NaHCO₃ and extracted 3× with DCM. Thecombined organic phases were dried over NaSO₄ and the solvent wasevaporated under reduced pressure.

Yield: 303 mg (1.13 mmol)

MS: m/z 269.3=[M+H]⁺ (MW calculated=268.4 g/mol)

Example 17 Synthesis of Linker 17a and 17b

Synthesis of 17a:

Fmoc-Asp(OtBu)-OH (322 mg, 0.78 mmol), Tmob-protected diamine 16 (150mg, 0.56 mmol), HATU (255 mg, 0.67 mmol) and DIEA (290 μL, 1.68 mmol)were dissolved in DMF (1.5 mL). The mixture was stirred for 30 min,acidified with AcOH and purified by RP-HPLC.

Yield: 463 mg (5.97 mmol, TFA salt, ca. 90% pure)

MS Fmoc-Asp(OtBu)-N(TMOB)CH₂CH₂N(CH₃)₂: m/z 662.5=[M+H]⁺ (MWcalculated=661.8 g/mol)

Fmoc-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂ (225 mg, 0.29 mmol) was dissolved ina solution of piperidine (50 μL) and DBU (15 μL) in DMF (1.5 mL). Themixture was stirred at RT for 1.5 h. AcOH was added andH-Asp(OtBu)-N(TMOB)CH₂CH₂N(CH₃)₂ was purified by RP-HPLC.

Yield: 114 mg (0.21 mmol, TFA salt)

MS H-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂: m/z 462.4=[M+Na]⁺ (MWcalculated=439.6 g/mol)

The TFA salt of H-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂ (114 mg, 0.21 mmol) wasdissolved in sat. NaHCO₃ (10 mL) and extracted 3× with DCM (3×10 mL).The combined organic layers were dried over NaSO₄ and the solvent wasremoved under reduced pressure. The residue was dissolved in DMF (1.0mL), 6-tritylmercaptohexanoic acid (121 mg, 0.31 mmol), HATU (118 mg,0.31 mmol) and DIEA (108 μL, 0.62 mmol) were added. The mixture wasstirred for 30 min AcOH was added (200 μL) andTrtS(CH₂)₅CONH-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂ was purified by RP-HPLC.

Yield: 95 mg (0.10 mmol, TFA salt)

MS TrtS(CH₂)₅CONH-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂: m/z 812.64=[M+H]⁺ (MWcalculated=812.1 g/mol)

TrtS(CH₂)₅CONH-Asp(OtBu)-N(Tmob)CH₂CH₂N(CH₃)₂ (95 mg, 0.10 mmol) wasdissolved in a 3:1 mixture of MeOH/H₂O (1.0 mL), LiOH (7.4 mg, 0.31mmol) was added and the mixture was stirred for 5 h at 60° C. AcOH wasadded (100 μL) and 17a was purified by RP-HPLC.

Yield: 64 mg (0.07 mmol, TFA salt)

MS 17a: m/z 756.5=[M+H]⁺ (MW calculated=756.0 g/mol)

17b was synthesized as described above except for the use ofFmoc-NMe-Asp(OtBu)-OH instead of Fmoc-Asp(OtBu)-OH in the first step.

Yield 17b: 16 mg (18 μmol, TFA salt)

MS 17b: m/z 770.5=[M+H]⁺ (MW calculated=770.0 g/mol)

Example 18 Synthesis of linker-BNP conjugates 18a and 18b

Synthesis of 18a:

17a (8.0 mg, 0.01 mmol), PyBOP (5.2 mg, 10 μmol) and DIEA (7 μL, 40μmol) were dissolved in DMF (400 μL) and immediately added to resinbound, side chain protected BNP-32a (50 mg, 5 μmol). After incubationfor 2 h at RT, the resin was washed with 10×DMF, 10×DCM and dried invacuo. The product was cleaved from the resin and purified by RP-HPLC.

Yield: 10.6 mg

MS 18a: m/z 930.4=[M+4H]⁴⁺; 1240.1=[M+3H]³⁺ (MW calculated=3717.2 g/mol)

18b was synthesized as described above except for the use of 17b insteadof 17a.

Yield: 4.7 mg

MS 18b: m/z 933.9=[M+4H]⁴⁺; 1244.7=[M+3H]³⁺ (MW calculated=3731.0 g/mol)

Example 19 Synthesis of PEG40 kDa-Linker-BNP Conjugates 19a and 19b

18a (5.2 mg) was dissolved in 1:1 H₂O/acetonitrile containing 0.1% TFA(200 μL). A solution of PEG40 kDa-maleimide (70 mg) in 1:1H₂O/acetonitrile (1.5 mL) and phosphate buffer (30 μL, pH 7.4, 0.5 M)was added. The solution was incubated at RT, after 5 min AcOH (30 μL)was added. 19a was purified by cation exchange chromatography, desalted,and lyophilized.

Yield: 19.2 mg

19b was synthesized as described for 19a except for the use of 18binstead of 18a.

Example 20 Synthesis of Linker 20

Fmoc-Asp(OH)OtBu (100 mg, 0.24 mmol), H₂N—(CH₂)₂—N(CH₃)-boc (36 μL, 0.20mmol), HATU (92 mg, 0.24 mmol) and DIEA (105 μL, 0.60 mmol) weredissolved in 1 mL DMF. The mixture was stirred for 1 h at RT, acidifiedwith AcOH (100 μL) and purified by HPLC.

Yield: 91 mg (0.13 mmol)

MS Fmoc-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu: 590.3=[M+Na]⁺ (MW calculated=567.7g/mol)

Fmoc-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu (91 mg, 0.13 mmol) was dissolved in DMF(1.0 mL), piperidine (50 μL) and DBU (15 μL) were added and the mixturewas stirred for 45 min at RT. AcOH (100 μL) was added andNH₂-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu was purified by RP-HPLC.

Yield: 39 mg (0.09 mmol, TFA salt)

MS NH₂-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu: m/z 368.1=[M+Na]⁺ (MWcalculated=345.4 g/mol)

NH₂-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu (36 mg, 0.09 mmol) was dissolved in DMF(0.5 mL), 6-tritylmercaptohexanoic acid (55 mg, 0.14 mmol), HATU (53 mg,0.14 mmol) and DIEA (49 μL, 0.28 mmol) were added. The mixture wasstirred for 45 min. AcOH was added (100 μL) andTrtS(CH₂)₅CONH-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu was purified by RP-HPLC.

Yield: 41 mg (0.06 mmol)

MS TrtS(CH₂)₅CONH-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu: m/z 740.6=[M+Na]⁺ (MWcalculated=718.0 g/mol)

TrtS(CH₂)₅CONH-Asp(NH(CH₂)₂N(CH₃)-boc)OtBu (41 mg, 0.06 mmol) wasdissolved in 1:1 dioxane/H₂O (1.0 mL), LiOH (4.1 mg, 0.17 mmol) wasadded and the mixture was stirred at 60° C. for 1 h. AcOH (50 μL) wasadded and 20 was purified by RP-HPLC.

Yield: 31 mg (0.05 mmol)

MS 20: m/z 684.5=[M+Na]⁺ (MW calculated=661.9 g/mol)

Example 21 Synthesis of Linker-Exendin Conjugate 21

Resin bound, side chain protected exendin (50 mg, 5 μmol) with a Mmtprotecting-group on Lys27 was first boc-protected at the N-terminus (seeMaterials and Methods) and then incubated five times (5 min) with 2 mLof 9/1 (v/v) DCM/HFIP to remove the Mmt protecting group from Lys27. 20(6.6 mg, 10 μmol), PyBOP (5.2 mg, 10 μmol and DIEA (7 μL, 40 μmol) weredissolved in DMF (400 μL) and immediately added to the resin. Incubationfor 3 h at RT, the resin was washed with 10×DMF, 10×DCM and dried invacuo. The product was cleaved from the resin and purified by RP-HPLC.

Yield: 2.4 mg

MS 21: m/z 1497.2=[M+3H]³⁺ (MW calculated=4488.0 g/mol)

Example 22 Synthesis of Fatty Acid-Linker-Exendin Conjugate 22

21 (2.6 mg) was dissolved in 200 μl 1/1 acetonitrile/water and 1 (0.8mg) in 400 μl of 7/3 acetonitrile/water was added. 100 μl of 0.25 Msodium phosphate buffer was added, the reaction was stirred for 5 minafter which 22 was purified by RP-HPLC.

Yield: 2.4 mg

MS 22: m/z 1388.3=[M+4H]⁴⁺; 1857.1=[M+3H]³⁺ (MW calculated=5566.4g/mol).

Example 23 Synthesis of Precursor 23

6-Tritylmercaptohexanoic acid (200 mg, 0.51 mmol), (PfpO)₂CO (202 mg,0.51 mmol) and collidine (340 μL, 2.65 mmol) were dissolved in DMSO (1mL) and stirred for 30 min at RT. The mixture was added to a solution ofFmoc-Lys-OH (170 mg, 0.46 mmol) in H₂O/pyridine/tBuOH (3:3:1, 6 mL). Thereaction was heated at 60° C. for 2 h, diluted with EtOAc, extracted 2×with 0.1 M H₂SO₄, 2× with brine and dried over Na₂SO₄. The solvent wasevaporated under reduced pressure and the residue was purified byRP-HPLC.

Yield: 109 mg

MS 23: m/z 741.3 [M+H]⁺ (MW calculated=741.0 g/mol)

Example 24 Synthesis of Linker 24a-24c

23 (186 mg, 0.25 mmol) and DIEA (160 μL, 0.92 mmol) were dissolved inDCM (2 mL), added to 2-chlorotrityl chloride resin (312 mg, 1.3 mmol/g)and agitated for 45 min at RT. MeOH (0.6 mL) was added and the resin wasincubated for another 15 min. The resin was washed with DCM (10×) andDMF (10×). Fmoc-deprotection and urea formation was achieved accordingto general procedures (see Materials and Methods) by reaction withN-boc-ethylenediamine (57 μL, 0.34 mmol), the product was cleaved fromthe resin and purified by RP-HPLC.

Yield: 14 mg

MS 24a: m/z 705.4 [M+H]⁺, 727.3 [M+Na]⁺ (MW calculated=704.9 g/mol)

24b was synthesized as described for 24a except for the use ofN-boc-N-methylethylenediamine instead of instead ofN-boc-ethylenediamine.

Yield: 21 mg

MS 24b: m/z 719.3 [M+H]⁺, 741.4 [M+Na]⁺ (MW calculated=719.0 g/mol)

24c was synthesized as described for 24a except for the use ofN,N-dimethylethylenediamine instead of N-boc-ethylenediamine.

Yield: 10 mg

MS 24c: m/z 633.2 [M+H]⁺ (MW calculated=632.9 g/mol)

Example 25 Synthesis of Exendin-Linker Conjugates 25a-25c

24a (7.0 mg, 0.01 mmol), PyBOP (5.2 mg, 10 μmol) and DIEA (7 μL, 40μmol) were dissolved in DMF (250 μL), immediately added to resin bound,side chain protected exendin (50 mg, 5 μmol) and incubated for 2 h atRT. The resin was washed with DMF (10×), DCM (10×) and dried in vacuo.The product was cleaved from the resin and purified by RP-HPLC.

Yield: 1.6 mg

MS 25a: m/z 1511.8=[M+3H]³⁺ (MW calculated=4530.8 g/mol)

25b was synthesized as described for 25a except for the use of 24binstead of 24a.

Yield: 4.3 mg

MS 25b: m/z 1516.3=[M+3H]³⁺ (MW calculated=4544.8 g/mol)

25c was synthesized as described for 25a except for the use of 24cinstead of 24a.

Yield: 1.3 mg

MS 25c: m/z 1520.4=[M+3H]³⁺ (MW calculated=4558.8 g/mol)

Example 26 Synthesis of Fatty Acid-Linker Conjugates 26a-26c

25a (1.6 mg) was dissolved in 200 μl 1/1 acetonitrile/water and 1 (0.11mg) in 200 μl of 7/3 acetonitrile/water was added. 30 μl of 0.25 Msodium phosphate buffer was added, the reaction was stirred for 5 min,after which 26a was purified by RP-HPLC.

MS 26a: m/z 1870.0=[M+3H]³⁺ (MW calculated=5609.2 g/mol).

26b was synthesized as described for 26a except for the use of 25binstead of 25a.

MS 26b: m/z 1875.9=[M+3H]³⁺, 1406.7=[M+4H]⁴⁺ (MW calculated=5623.2g/mol)

26c was synthesized as described for 26a except for the use of 25cinstead of 25a.

MS 26c: m/z 1879.4=[M+3H]³⁺, 1410.5=[M+4H]⁴⁺ (MW calculated=5637.2g/mol)

Example 27 Synthesis of 20KDa-PEG-Linker-Exendin Conjugates 27a-27c

25a (2.0 mg) was dissolved in 1:1 H₂O/MeCN containing 0.1% TFA (200 μl).A solution of PEG40 kDa-maleimide (18 mg) in 1:1 H₂O/MeCN (1 ml) andphosphate buffer (15 μl, pH 7.4, 0.5 M) was added. The solution wasincubated at RT, after 5 min AcOH (20 μl) was added and 27a was purifiedby cation exchange chromatography, desalted and lyophilized.

27b was synthesized as described for 27a except for the use of 25binstead of 25a.

27c was synthesized as described for 27a except for the use of 25cinstead of 25a.

Example 28 Synthesis of Linker 28

6-Bromohexanoyl chloride (46 μl, 0.31 mmol) was dissolved in 0.2 mlCH₂Cl₂ and added to a solution of H₂N—CH₂—CH₂—STrt (100 mg, 0.28 mmol),DIEA (97 μl, 0.56 mmol) in CH₂Cl₂ (0.8 ml). The mixture was stirred for2 h at RT. The reaction mixture was acidified with AcOH (50 μl) and thesolvent was removed in vacuo. The residue was purified on silica gel(heptane/EtOAc=1:1) to obtain Br—(CH₂)₅—CONH—(CH₂)₂—STrt.

Yield: 137 mg (0.276 mmol, 98%)

MS Br—(CH₂)₅—CONH—(CH₂)₂—STrt: 518.9=[M+Na]⁺, (MW calculated=496.5g/mol)

N-Boc-ethylenediamine (81 μl, 0.51 mmol) was added to a solution ofBr—(CH₂)₅—CONH—(CH₂)₂-STrt (230 mg, 0.46 mmol) and Na₂CO₃ (196 mg, 1.85mmol) in DMF (0.8 ml). The reaction mixture was stirred for 10 h at 70°C. After cooling to RT the mixture was diluted with 4 ml(MeCN/H₂O=25:75, with 0.1% TFA) and purified by RP-HPLC to getBoc-NH—(CH₂)₂—NH—(CH₂)₅—CONH—(CH₂)₂—STrt.

Yield: 189 mg (0.27 mmol, 59%, TFA-salt)

MS Boc-NH—(CH₂)₂—NH—(CH₂)₅—CONH—(CH₂)₂—STrt: 576.5=[M+H]⁺, (MWcalculated=575.5 g/mol)

Boc-NH—(CH₂)₂—NH—(CH₂)₅—CONH—(CH₂)₂—STrt (189 mg, 0.27 mmol) and HCHO(35% aqueous, 113 μl) were dissolved in MeCN (1.5 ml) and NaCNBH₃ (34mg, 0.54 mmol) was added. The reaction mixture was stirred for 5 h atRT. After completion of the reaction (MS) the solution was diluted withH₂O (5 ml) and extracted with CH₂Cl₂ (3×5 ml). The combined organiclayers were dried over MgSO₄, filtered and the solvent was removed invacuo. The residue was purified by RP-HPLC to getBoc-NH—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂—STrt.

Yield: 62.8 mg (0.11 mmol, 39%)

MS Boc-NH—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂—STrt: 590.6=[M+H]⁺, (MWcalculated=589.0 g/mol)

Boc-NH—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂—STrt (62.8 mg, 0.11 mmol) wasdissolved in THF (6 ml) and HCl in dioxane (130 μl, 4 M solution) wasadded. The reaction mixture was stirred for 12 h at RT. 200 μl HCl indioxane was added and the solvent was removed in vacuo. The residue waspurified by RP-HPLC to give H₂N—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂-STrtand not consumed starting materialBoc-NH—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂-STrt.

Yield: 32.8 mg (0.062 mmol, 44%, HCl-salt) 28 and 14.7 mg (0.025 mmol,23%, TFA-salt) starting material

MS 28: 490.5=[M+H]⁺, (MW calculated=489.0 g/mol)

Example 29 General Procedure for the Synthesis Carboxylic AcidSubstituted SNP Precursors 29a and 29b

Cis-cyclohexane-1,2-dicarboxylic anhydride (231 mg, 1.5 mmol) andpyridine (271 μl, 2 mmol) were dissolved in DCM (2 ml) and added toresin bound, side chain protected BNP-32a (300 mg). Incubation for 1 hat RT, washed with 10×DCM and dried in vacuo.

Resin bound, side chain protected BNP-32b, carrying an ivDde protectinggroup at Lys14, was first boc-protected at the N-terminus, deprotectedat Lys14 position (see Materials and Methods) and then reacted withcis-cyclohexane-1,2-dicarboxylic anhydride as described above for 29a.

Example 30 Synthesis of BNP-Linker-Thiols 30a and 30b

H₂N—(CH₂)₂—N(CH₃)—(CH₂)₅—CONH—(CH₂)₂—STrt 28 (5.2 mg, 0.01 mmol), PyBOP(5.2 mg, 0.01 mmol) and DIEA (7.0 μl, 0.04 mmol) were dissolved in DMF(300 μl) and added to resin bound, side chain protected BNP 29a (50 mg,0.005 mmol). Incubation for 2 h at RT, the resin was washed with DMF(10×), DCM (10×) and dried in vacuo. The product was cleaved from theresin and purified by RP-HPLC.

Yield: 9.8 mg

MS 30a: m/z 947.6=[M+4H]⁴⁺, 1263.1=[M+3H]³ (MW calculated=3786.3 g/mol)

30b was synthesized as described above except for the use of resin boundBNP derivative 29b instead of 29a.

Yield: 7.4 mg

MS 30b: m/z 947.5=[M+4H]⁴⁺, 1263.0=[M+3H]³⁺ (MW calculated=3786.3 g/mol)

Example 31 Synthesis of 40KDa-PEG-Linker-BNP Conjugates 31a and 31b

30a (4 mg) was dissolved in 1:1 H₂O/MeCN containing 0.1% TFA (200 μl). Asolution of PEG40KDa-maleimide (42.2 mg) in 1:1 H₂O/MeCN (1 ml) andphosphate buffer (15 μl, pH 7.4, 0.5 M) was added. The solution wasincubated at RT, after 5 min AcOH (20 μl) was added and 31a was purifiedby cation exchange chromatography, desalted and lyophilized.

Yield: 2.0 mg

31b was synthesized as described for 31a except for the use of 30binstead of 30a.

Yield: 16.8 mg

Example 32 Synthesis of Linker 32

3-Bromopropionyl chloride (62.5 μl, 0.62 mmol) was dissolved in 0.5 mlCH₂Cl₂ and added to a solution of H₂N—CH₂—CH₂—STrt (200 mg, 0.56 mmol),DIEA (196 μl, 1.1 mmol) in CH₂Cl₂ (1 ml). The mixture was stirred for 1h at RT. The reaction mixture was acidified with AcOH (100 μl) and thesolvent was removed in vacuo. The residue was purified over silica gel(heptane/EtOAc=1:1) to obtain Br—(CH₂)₂CONH—(CH₂)₂—STrt.

Yield: 223 mg (0.49 mmol, 87%)

MS Br—(CH₂)₂CONH—(CH₂)₂—STrt: 478.7=[M+Na]⁺, (MW calculated=454.7 g/mol)

N-Alloc-ethylenediamine HCl-salt (43.5 mg, 0.24 mmol) and DIEA (38 μl,0.22 mmol) were added to a solution of Br—(CH₂)₂CONH—(CH₂)—STrt (100 mg,0.22 mmol) and Na₂CO₃ (93 mg, 0.87 mmol) in DMF (1 ml). The reactionmixture was stirred for 10 h at 70° C. After cooling down to RT thereaction mixture was diluted with 4 ml (MeCN/H₂O=25:75, with 0.1% TFA)and purified by HPLC to get Alloc-NH—(CH₂)₂—NH—(CH₂)₂—CONH—(CH₂)₂—STrt.

Yield: 61 mg (0.096 mmol, 44%, TFA salt)

MS Alloc-NH—(CH₂)₂—NH—(CH₂)₂—CONH—(CH₂)₂—STrt: 540.8=[M+Na]⁴, (MWcalculated=517.8. g/mol)

Alloc-NH—(CH₂)₂—NH—(CH₂)₂—CONH—(CH₂)₂—STrt (60.9 mg, 0.096 mmol) wasdissolved in CH₂Cl₂ and Boc₂O (42 mg, 0.19 mmol) was added. The solutionwas stirred for 20 h at RT. After completion the reaction was quenchedby addition of 70 μl AcOH and the solvent was removed in vacuo. Theresidue was diluted with 4 ml MeCN/H₂O (25:75, with 0.1% TFA) andpurified by RP-HPLC to giveAlloc-NH—(CH₂)₂—N(Boc)-(CH₂)₂—CONH—(CH₂)₂—STrt.

Yield: 53.3 mg (0.086 mmol, 89%)

MS Alloc-NH—(CH₂)₂—N(Boc)-(CH₂)₂—CONH—(CH₂)₂—STrt: 640.6=[M+Na]⁺, (MWcalculated=617.9. g/mol)

Alloc-NH—(CH₂)₂—N(Boc)-(CH₂)₂—CONH—(CH₂)₂—STrt (48.3 mg, 0.078 mmol) wasdissolved in THF, triethylammonium format (62 μl) and Pd(PPh₃)₄ (16 mg)were added. The solution was stirred for 12 h at RT and monitored by MS.After completion the solvent was removed in vacuo. The residue wasdissolved in MeCN/H₂O (50:50, with 0.1% TFA) and purified by RP-HPLC togive H₂N—(CH₂)₂—N(Boc)-(CH₂)₂—CONH—(CH₂)₂—STrt (31).

Yield: 20.1 mg (0.031 mmol, 40%, TBA-salt)

MS 31: 534.6=[M+H]⁺, 556.6=[M+Na]⁺, (MW calculated=533.5 g/mol)

Example 33 Synthesis of BNP-Linker-Thiols 33a and 33b

33a was synthesized as described for 30a except for the use of 32instead of 28.

Yield: 8.0 mg

MS 33a: m/z 933.5=[M+4H]⁴⁺, 1244.3=[M+3H]³⁺ (MW calculated=3729.9 g/mol)

33b was synthesized as described for 30b except for the use of 32instead of 28.

Yield: 5.0 mg

MS 33b: m/z 933.5=[M+4H]⁴⁺, 1244.3=[M+3H]³⁺ (MW calculated=3715.9 g/mol)

Example 34 Synthesis of 40KDa-PEG-Linker-BNP Conjugates 34a and 34b

33a (4.3 mg) was dissolved in 1:1 H₂O/MeCN containing 0.1% TFA (200 μl).A solution of PEG40KDa-maleimide (46.8 mg) in 1:1 H₂O/MeCN (1 ml) andphosphate buffer (20 μl, pH 7.4, 0.5 M) was added. The solution wasincubated at RT, after 5 min AcOH (20 μl) was added and 34a was purifiedby cation exchange chromatography, desalted and lyophilized.

Yield: 9.7 mg

34b was synthesized as described for 34a except for the use of 33binstead of 33a.

Yield: 11.5 mg

Example 35 Synthesis of Linker 35

Bromoacetylbromide (54 μl, 0.62 mmol) was dissolved in 0.5 ml CH₂Cl₂ andadded to a solution of H₂N—CH₂—CH₂—STrt (200 mg, 0.56 mmol) and DIEA(196 μl, 1.1 mmol) in CH₂Cl₂ (1 ml). The mixture was stirred for 1 h atRT. The reaction mixture was acidified with AcOH (100 μl) and thesolvent was removed in vacuo. The residue was purified over silica gel(heptane/EtOAc=1:1) to obtain product Br—CH₂—CONH—(CH₂)₂—STrt.

Yield: 245 mg (0.55 mmol, 99%)

MS Br—CH₂—CONH—(CH₂)₂—STrt: 462.4=[M+Na]⁺, (MW calculated=440.4 g/mol)

N-Alloc-ethylenediamine HCl-salt (45 mg, 0.25 mmol) and DIEA (79 μl,0.45 mmol) was added to a solution of Br—CH₂—CONH—(CH₂)₂—STrt (100 mg,0.23 mmol) in DMF (1 ml). The reaction mixture was stirred for 10 h at70° C. After cooling down to RT the reaction mixture was diluted withH₂O/Et₂O (1:1, 40 ml) and the layers were separated. The aqueous layerwas extracted several times with Et₂O. The combined organic layers weredried with MgSO₄, filtered and the solvent was removed in vacuo. Theresidue was purified over silica gel (DCM/MeOH=95:5) to giveAlloc-NH—(CH₂)₂—NH—CH₂—CONH—(CH₂)₂—STrt.

Yield: 94 mg (0.186 mmol, 82%, contains residual DMF)

MS Alloc-NH—(CH₂)₂—NH—CH₂—CONH—(CH₂)₂—STrt: 526.8=[M+Na]⁺, (MWcalculated=503.8. g/mol)

Alloc-NH—(CH₂)₂—NH—CH₂—CONH—(CH₂)₂—STrt (94 mg, 0.186 mmol, with DMF))was dissolved in CH₂Cl₂ and Boc₂O (81 mg, 0.37 mmol) was added. Thesolution was stirred for 20 h at RT. After completion the reaction wasquenched by addition of 100 μl AcOH and the solvent was removed invacuo. The residue was diluted with 4 ml MeCN/H₂O (25:75, with 0.1% TFA)and purified by RP-HPLC to giveAlloc-NH—(CH₂)₂—N(Boc)-CH₂—CONH—(CH₂)₂—STrt.

Yield: 34.7 mg (0.057 mmol, 26%)

MS Alloc-NH—(CH₂)₂—N(Boc)-CH₂—CONH—(CH₂)₂—STrt: 603.9=[M+Na]⁺, (MWcalculated=603.9. g/mol)

Alloc-NH—(CH₂)₂—N(Boc)-CH₂—CONH—(CH₂)₂—STrt (34.7 mg, 0.048 mmol) wasdissolved in THF, triethylammoniumformate (38 μl) and Pd(PPh₃)₄ (5 mg)were added. The solution was stirred for 12 h at RT and monitored by MS.After completion the solvent was removed in vacuo. The residue wasdissolved in MeCN/H₂O (50:50, with 0.1% TFA) and purified by RP-HPLC togive H₂N—(CH₂)₂—N(Boc)-CH₂—CONH—(CH₂)₂—STrt 35.

Yield: 12.6 mg (0.019 mmol, 42%, TFA-salt)

MS 35: 520.1=[M+H]⁺, 542.2=[M+Na], (MW calculated=519.2 g/mol)

Example 36 Synthesis of BNP-Linker-Thiols 36a and 36b

36a was synthesized as described for 30a except for the use of 35instead of 28.

Yield: 9.1 mg

MS 36a: m/z 930.0=[M+4H]⁴⁺, 1239.6=[M+3H]³⁺ (MW calculated=3715.9 g/mol)

36b was synthesized as described for 30b except for the use of 35instead of 28.

Yield: 8.0 mg

MS 36b: m/z 929.9=[M+4H]⁴⁺, 1239.5=[M+3H]³⁺ (MW calculated=3715.9 g/mol)

Example 37 Synthesis of 40KDa-PEG-Linker-BNP Conjugates 37a and 37b

36a (4.2 mg) was dissolved in 1:1 H₂O/MeCN containing 0.1% TFA (200 μl).A solution of PEG40KDa-maleimide (68 mg) in 1:1 H₂O/MeCN (1 ml) andphosphate buffer (20 pH 7.4, 0.5 M) was added. The solution wasincubated at RT, after 5 min AcOH (20 μl) was added and 37a was purifiedby ion exchange chromatography, desalted and lyophilized

Yield: 16 mg

37b was synthesized as described for 37a except for the use of 36binstead of 36a.

Yield: 18.5 mg

Example 38 Synthesis of Linker-Exendin Conjugates

Linker-exendin conjugates were synthesized according to generalsynthesis method A, B, C, D, E or F.

Method a

Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2 mmol) weredissolved in 0.3 ml of dry DMF. Mixture was added to side chainprotected exendin-4 on resin (2 μmol) and agitated for 30 min at roomtemperature. Resin was washed with DMF (10 times). PyBOP (0.1 mmol) anddiamine (0.1 mmol) were dissolved in 0.3 ml of dry DMF. Mixture wasadded to resin and agitated for 30 min at room temperature. Resin waswashed with DMF (10 times). Exendin-linker conjugates were cleaved andpurified by RP-HPLC as described in “Materials and Methods”.

Method B

Synthesis: As described for Method A except that diacid anhydride andpyridine are replaced by diacid (0.2 mmol), HOBt (0.2 mmol), DIC (0.2mmol), and collidine (0.4 mmol).

Method C

Synthesis. Diamine (0.6 mmol) was dissolved in 1 ml of dry DCM anddiacid anhydride (0.4 mmol) was added. Mixture was stirred for 60 min atroom temperature. DCM was removed, the residue was dissolved inACN/water/AcOH, and amino acid was purified by RP-HPLC and lyophilized.

Amino acid (0.1 mmol), HOBt (0.1 mmol), DIC (0.1 mmol), and collidine(0.2 mmol) were dissolved in 0.3 ml of dry DMF. Mixture was added toexendin-4 on resin (2 μmol) and agitated for 30 min at room temperature.Resin was washed with DMF (10 times). Exendin-linker conjugates werecleaved and purified by RP-HPLC as described in “Materials and Methods”.

Method D

Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2 mmol) weredissolved in 0.3 ml of dry DMF. Mixture was added to exendin-4 on resin(2 μmol) and agitated for 30 min at room temperature. Resin was washedwith DMF (10 times). PyBOP (0.1 mmol), HOBt (0.1 mmol), and collidine(0.4 mmol) were dissolved in 0.3 ml of dry DMF. Mixture was added toresin and agitated for 30 min at room temperature. Resin was washed withDMF (10 times). Diamine (0.1 mmol) and DIEA (0.3 mmol) were dissolved ina mixture of 0.4 ml of DMF and 0.4 ml of EtOH. Mixture was added toresin and agitated for 30 min at room temperature. Resin was washed withDMF (10 times).

Exendin-linker conjugates were cleaved and purified by RP-HPLC asdescribed in “Materials and Methods”.

Method E

Synthesis: Fmoc amino acid (0.1 mmol), PyBOP (0.1 mmol) and DIEA (0.2mmol) were dissolved in 0.3 ml of dry DMF. Mixture was added toexendin-4 on resin (2 μmol) and agitated for 30 min at room temperature.Fmoc protecting group was removed by incubating resin in DMF/piperidine4/1 (v/v) for 2×10 min. Resin was washed with DMF (10 times) and DCM (10times). p-Nitrophenyl chloroformate (0.1 mmol) was dissolved in 0.3 mlof dry THF and DIEA (0.2 mmol). Mixture was added to resin and agitatedfor 30 min at room temperature. Resin was washed with DCM (10 times).Diamine (0.1 mmol) was dissolved in 0.3 ml of DMF. Mixture was added toresin and agitated for 30 min at room temperature. Resin was washed withDMF (10 times).

Exendin-linker conjugates were cleaved and purified by RP-HPLC asdescribed in “Materials and Methods”.

Method F

Synthesis: as described for Method A, followed by fmoc-deprotection andacetylation: Fmoc protecting group was removed as by incubating resin inDMF/piperidine 4/1 (v/v) for 2×10 min. Resin was washed with DMF (10times). Acetylation was performed by incubating resin with aceticanhydride/pyridine/DMF 1/1/2 (v/v/v) for 30 min. Resin was washed withDMF (10 times).

Exendin-linker conjugates were cleaved and purified by RP-HPLC asdescribed in “Materials and Methods”.

Further details concerning compound numerals, starting materials,synthesis method, molecular weight (MW) and MS data are given in FIG. 2.

Example 39 Synthesis of Hydrogel-Linker-Exendin Conjugates 39

Maleimide-functionalized hydrogel microparticles were synthesized asdescribed in EP 1 625 856 A1.

30 mg of maleimide-derivatized hydrogel microparticles (loading 40μmol/g, 1.2 μmol) were reacted with 6 mg of compound 25a (1.32 μmol, 1.1eq) in 600 μl 20/80 (v/v) acetonitrile/50 mM phosphate buffer (pH 7.4)for 10 min to give exendin-linker loaded hydrogel microparticles 39. Theloaded hydrogel 39 was washed 5 times with 50/50 (v/v)acetonitrile/water and three times with water.

Example 40 Synthesis of Linker 40

Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 μL, 1.0 mmol) weredissolved in DCM (2 mL), added to 2-chlorotrityl chloride resin (312 mg,1.3 mmol/g) and agitated for 45 min at RT. Methanol (0.6 mL) was addedand the resin was incubated for another 15 min. The resin was washedwith DCM (10×) and DMF (10×). Fmoc-deprotection and urea formation wasachieved according to general procedures (see Materials and Methods) byreaction with linker intermediate 5a, the product was cleaved from theresin and purified by RP-HPLC.

Yield: 53 mg

MS 40: m/z 604.4 [M+H]⁺ (MW calculated=603.8 g/mol)

Example 41 Synthesis of Exendin-Linker Conjugate 41

40 (HCl salt, 14.0 mg, 0.02 mmol), PyBOP (10.2 mg, 0.02 mmol) and DIEA(17 μL, 0.1 mmol) were dissolved in DMF (300 μL), immediately added toresin bound, side chain protected exendin (100 mg, 10 μmol) andincubated for 4 h at RT. The resin was washed with DMF (10×), DCM (10×)and dried in vacuo. The product was cleaved from the resin and purifiedby RP-HPLC.

Yield: 5.4 mg

MS 40: m/z 1510.9=[M+3H]³⁺ (MW calculated=4530.1 g/mol)

Example 42 Synthesis of Fatty Acid-Linker Conjugate 42

41 (1.6 mg) was dissolved in 200 μl 3/l acetonitrile/water and 1 (0.11mg) in 200 μl of 3/l acetonitrile/water was added. 30 μl of 0.25 Msodium phosphate buffer was added, the reaction was stirred for 5 min,after which 42 was purified by RP-HPLC.

MS 42: m/z 1870.2=[M+3H]³⁺ (MW calculated=5608.4 g/mol).

Example 43 Synthesis of Hydrogel-Linker-Exendin Conjugate 43

43 was synthesized as described for 39 except for the use of 41 insteadof 25a.

Example 44 Synthesis of Linker 44

44 was synthesized as described for 40 except for the use of 28 insteadof 5a.

Yield: 74 mg

MS 44: m/z 605.4 [M+H]⁺ (MW calculated=604.8 g/mol)

Example 45 Synthesis of Exendin-Linker Conjugate 45

45 was synthesized as described for 41 except for the use of 44 insteadof 40.

Yield: 6.0 mg

MS 45: m/z 1511.3=[M+3H]³⁺ (MW calculated=4531.1 g/mol)

Example 46 Synthesis of fatty acid-linker conjugate 46

46 was synthesized as described for 42 except for the use of 45 insteadof 41.

MS 46: m/z 1870.5=[M+3H]³⁺ (MW calculated=5609.5 g/mol).

Example 47 Synthesis of Linker Intermediate 47

Trityl sulfide (247 mg, 0.89 mmol) was suspended in 1 ml DMSO. DBU (152μl, 1.02 mmol) and 6-bromohexan-1-ol (173 mg, 0.96) were added andmixture was stirred for 5 min at RT. Reaction mixture was dissolved in20 ml ethylacetate and washed with 1 N H₂SO₄ (2×) and brine (3×).Organic layer was dried (Na₂SO₄) and volatiles were removed in vacuo.Product was purified by flash chromatography on silica (heptane/AcOEt1/l).

Yield 283 mg (S-trityl)-6-mercaptohexan-1-ol

(S-Trityl)-6-mercaptohexan-1-ol (466 mg, 1.24 mmol) was dissolved in 3.5ml DCM, 0.5 ml DMSO and 0.6 ml NEt₃, and cooled in an ice bath.SO₃-pyridine (408 mg, 2.57 mmol) was suspended in 0.5 ml DMSO and addedto reaction mixture. Ice bath was removed and reaction was stirred for60 min at RT. Reaction mixture was dissolved in 20 ml Et₂O and extractedwith 1 N H₂SO₄ (2×) and brine (3×). Organic layer was dried (Na₂SO₄) andvolatiles were removed in vacuo. Product was purified by flashchromatography on silica (heptane/AcOEt 1/l).

Yield: 390 mg (S-trityl)-6-mercaptohexan-1-al 47

MS 47: m/z 243.1=[Trt]⁺, 413.1=[M+K]⁺ (MW calculated=374.4 g/mol)

Example 48 Synthesis of Linker 48

Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 μL, 1.0 mmol) weredissolved in DCM (2 mL), added to 2-chlorotrityl chloride resin (312 mg,1.3 mmol/g) and agitated for 45 min at RT. Methanol (0.6 mL) was addedand the resin was incubated for another 15 min. The resin was washedwith DCM (10×) and DMF (10×). Fmoc-deprotection and urea formation wasachieved according to general procedures (see Materials and Methods) byreaction with ethylene diamine. For reductive alkylation 47 (299 mg, 0.8mmol) and Na(OAc)₃BH (340 mg, 1.6 mmol) were dissolved in 0.5 mL DMF,0.5 ml MeOH and 10 μL AcOH, added to resin and agitated for 2 h at RT.Resin was washed with DMF (10×) and DCM (10×). Boc protection wasperformed by agitating resin in a solution of boc anhydride (218 mg, 1.0mmol) and DIEA (170 μL, 1.0 mmol) in DCM. Resin was washed with DCM(10×) and product was cleaved from the resin and purified by RP-HPLC.

Yield: 34 mg

MS 48: m/z 634.2 [M+H]⁺ (MW calculated=633.9 g/mol)

Example 49 Synthesis of Exendin-Linker Conjugate 49

49 was synthesized as described for 41 except for the use of 48 insteadof 40.

Yield: 4.8 mg

MS 49: m/z 1487.3=[M+3H]³⁺ (MW calculated=4460.0 g/mol)

Example 50 Synthesis of Hydrogel-Linker-Exendin Conjugate 50

50 was synthesized as described for 39 except for the use of 49 insteadof 25a.

Example 51 Release Kinetics In Vitro

Release of drug molecule from 38a to 38z, 38aa to 38ab, 4, 5, 9a, 9b,9c, 10, 13a, 15, 19a, 19b, 22, 26a to 26c, 31a, 31b, 34a, 34b, 37a, 37b,42, 43, 46, and 50 was effected by hydrolysis in buffer at pH 7.4 and37° C. or pH 4 and 37° C. as described in “Materials and Methods”.

t_(1/2) buffer A t_(1/2) buffer B Compound (pH 7.4) (pH 4.0) 38a <1 h 13h 38b 20 h 72 d 38c >3 m >3 m 38d 58 d n.d. 38e 41 d n.d. 38f 23 h 114 d38g 19 d none 38h 47 d none 38i 69 h 108 d 38j 16 d n.d. 38k 40 min 6 d38l 16 h n.d. 38m 17 h 66 d 38n 18 d n.d. 38o 11-12 h 22 d 38p 26 d 178d 38q 26 d 210 d 38r 26 h 47 d 38s 80 min 80 h 38t 96 min 67 h 38u 51 dnone 38v 47 d none 38w 8 d 3.2 a 38x 72 d n.d. 38y 11-14 h 105 d 38z 11d 1.6 a 38aa 40 h 65 d 38ab 20 h 20 d 38ac 14 h n.d. 38ad 18 h n.d.  415 d n.d.  5 22 h n.d.  9a 340 h n.d  9b 360 h n.d  9c 120 h n.d 10 130h n.d 13a 120 h n.d 13b 160 h n.d. 15 160 h n.d 19a 31 h n.d. 19b 18 hn.d. 22 40 d n.d. 26a 34 d n.d. 26b 40 d n.d. 26c 18 d n.d. 31a 22 hn.d. 31b 95 h n.d. 34a 42 h n.d. 34b 205 h n.d. 37a 138 h n.d. 37b 639 hn.d. 42 10 d n.d. 43 17 d n.d. 46 13 d n.d. 50 35 d n.d.

Example 52 Release Kinetics In Vivo In Vitro/In Vivo Correlation

Release kinetics in vivo were determined by comparing thepharmacokinetics of 13a with the pharmacokinetics of 13c and 13b with13d, respectively, after intravenous injection into rat. Animal studieswere performed at Heidelberg Pharma AG, Heidelberg, Germany.

13a (27 mg) was dissolved in 3.5 ml PBS and 500 μl of the resultingsolution were injected intravenously into six rats each. Male SD ratswith approximately 270 g weight were used. Blood samples were drawn att=0, 2 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120 h, and 168 h, plasma wasprepared, and plasma was analyzed for fluorescein fluorescence using aPerkin-Elmer LS 50B spektrometer.

Pharmacokinetics of 13c were determined as described for 13a.Pharmacokinetics of 13b and 13d were determined as described for 13a,except for the use of 20 mg 13 b and 13d each in 2.5 ml PBS and fourrats.

Linker hydrolysis half-life was calculated from the ratio offluorescence of 13a compared to fluorescence of 13c and 13b compared to13d, respectively, at the respective time points.

Half-life of in vivo linker hydrolysis was determined to be 115 h and160 h for 13a and 13b, respectively, which is in excellent correlationto the half-life of in vitro linker hydrolysis of 120 h and 160 h for13a and 13b, respectively.

FIG. 3 shows in vivo and in vitro linker cleavage data of 13b, whereinin vivo (triangles) and in vitro (diamonds) cleavage kinetics are shownby semilogarithmic representation.

Abbreviations:

-   Acp 4-(2-aminoethyl)-1-carboxymethyl-piperazine-   AcOH acetic acid-   Boc t-butyloxycarbonyl-   Dab 2,4-diaminobutyric acid-   DBU 1,3-diazabicyclo[5.4.0]undecene-   DCM dichloromethane-   Dda dodecanoic acid-   DIC diisopropylcarbodiimide-   DIEA diisopropylethylamine-   DMAP dimethylamino-pyridine-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   EDTA ethylenediaminetetraacetic acid-   eq stoichiometric equivalent-   Fmoc 9-fluorenylmethoxycarbonyl-   HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HFIP hexafluoroisopropanol-   HEPES N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)-   HOBt N-hydroxybenzotriazole-   ivDde 1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methylbutyl-   LCMS mass spectrometry-coupled liquid chromatography-   Mal 3-maleimido propionyl-   Mmt 4-methoxytrityl-   MS mass spectrum-   MW molecular mass-   n.d. not determined-   PfpOH pentafluorophenol-   PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate-   RP-HPLC reversed-phase high performance liquid chromatography-   RT room temperature-   SEC size exclusion chromatography-   Suc succinimidopropionyl-   TCP 2-chlorotrityl chloride resin-   TES triethylsilane-   TMOB 2,4,6-trimethoxybenzyl-   TFA trifluoroacetic acid-   THF tetrahydrofurane-   UV ultraviolet-   VIS visual

1. A prodrug or a pharmaceutically acceptable salt thereof comprising: acleavable drug-linker conjugate D-L, which is configured so that thebond between the moiety D and the moiety L is cleaved after thedrug-linker conjugate D-L is administered so as to release a drug D-H;wherein: -D is a nitrogen containing biologically active moiety selectedfrom the group consisting of: antibody fragments, single chain antigenbinding proteins, catalytic antibodies, and fusion proteins; -L is anon-biologically active linker moiety -L¹; and -L¹ comprises anamine-containing nucleophile, and is represented by formula (I):

wherein: the dashed line indicates the attachment to the nitrogen of thebiologically active moiety by forming an amide bond; X is C(R⁴R^(4a)),N(R⁴), O, C(R⁴R^(4a))—C(R⁵R^(5a)), C(R⁵R^(5a))—C(R⁴R^(4a)),C(R⁴R^(4a))—N(R⁶), N(R⁶)—C(R⁴R^(4a)), C(R⁴R^(4a))—O, or O—C(R⁴R^(4a));X¹ is C, or S(O); X² is C(R⁷R^(7a)), or C(R⁷R^(7a))—C(R⁸R^(8a)); X³ isO, S, or N—CN; R¹, R^(1a), R², R^(2a), R³, R^(3a), R⁴, R^(4a), R⁵,R^(5a), R⁶, R⁷, R^(7a), R⁸, and R^(8a) are independently selected fromthe group consisting of H, and C₁₋₄ alkyl; optionally, one or more ofthe pairs R^(1a)/R^(4a), R^(1a)/R^(5a), R^(4a)/R^(5a), and R^(7a)/R^(8a)form a chemical bond; optionally, one or more of the pairs R¹/R^(1a),R²/R^(2a), R⁴/R^(4a), R⁵/R^(5a), R⁷/R^(7a), and R⁸/R^(8a) are joinedtogether with the atom to which they are attached to form a C₃₋₇cycloalkyl, or 4 to 7 membered heterocyclyl; optionally, one or more ofthe pairs R¹/R⁴, R¹/R⁵, R¹/R⁶, R⁴/R⁵, R⁴/R⁶, R⁷/R⁸, and R²/R³ are joinedtogether with the atoms to which they are attached to form a ring A;optionally, R³/R^(3a) are joined together with the nitrogen atom towhich they are attached to form a 4 to 7 membered heterocycle; A isselected from the group consisting of:  phenyl, naphthyl, indenyl,indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4 to 7 membered heterocyclyl, and9 to 11 membered heterobicyclyl; and —N(R³R^(3a)) is theamine-containing nucleophile; wherein L¹ is substituted with one to fourgroups L²-Z and optionally further substituted, provided that thehydrogen marked with the asterisk in formula (I) is not replaced by asubstituent, and wherein one or more further optional substituents areindependently selected from the group consisting of: halogen, CN, COOR⁹,OR⁹, C(O)R⁹, C(O)N(R⁹R^(9a)), S(O)₂N(R^(9a)), S(O)N(R⁹R^(9a)), S(O)₂R⁹,S(O)R⁹, N(R⁹)S(O)₂N(R^(9a)R^(9b)), SR⁹, N(R⁹R^(9a)), NO₂, OC(O)R⁹,N(R⁹)C(O)R^(9a), N(R⁹)S(O)₂R^(9a), N(R⁹)S(O)R^(9a), N(R⁹)C(O)OR^(9a),N(R⁹)C(O)N(R^(9a)R^(9b)), OC(O)N(R⁹R^(9a)), T, C₁₋₅₀alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl; wherein T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, andC₂₋₅₀ alkynyl are optionally substituted with one or more R¹⁰, which arethe same or different; wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of: T, —C(O)O—, —O—, —C(O)—, —C(O)N(R¹¹)—,—S(O)₂N(R¹¹)—, —S(O)N(R¹¹)—, —S(O)₂—, —S(O)—, —N(R¹¹)S(O)₂N(R^(11a))—,—S—, —N(R¹¹)—, —OC(O)R¹¹, —N(R¹¹)C(O)—, —N(R¹¹)S(O)₂—, —N(R¹¹)S(O)—,—N(R¹¹)C(O)O—, —N(R¹¹)C(O)N(R^(11a))—, and —OC(O)N(R¹¹R^(11a)); whereinR⁹, R^(9a), R^(9b) are independently selected from the group consistingof: H, T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein T,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different; and wherein C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of:  T, —C(O)O—,—O—, —C(O)—, —C(O)N(R¹¹)—, —S(O)₂N(R¹¹)—, —S(O)N(R¹¹)—, —S(O)₂—, —S(O)—,—N(R¹¹)S(O)₂N(R^(11a))—, —S—, —N(R¹¹)—, —OC(O)R¹¹, —N(R¹¹)C(O)—,—N(R¹¹)S(O)₂—, —N(R¹¹)S(O)—, —N(R¹¹)C(O)O—, —N(R¹¹)C(O)N(R^(11a))—, and—OC(O)N(R¹¹R^(11a)); wherein T is selected from the group consisting of:phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4 to 7membered heterocyclyl, and 9 to 11 membered heterobicyclyl; wherein T isoptionally substituted with one or more R¹⁰, which are the same ordifferent; wherein R¹⁰ is: halogen, CN, oxo (═O), COOR¹², OR¹², C(O)R¹²,C(O)N(R¹²R^(12a)), S(O)₂N(R¹²R^(12a)), S(O)N(R¹²R^(12a)), S(O)₂R¹²,S(O)R¹², N(R¹²)S(O)₂N(R^(12a)R^(12b)), SR¹², N(R¹²R^(12a)), NO₂,OC(O)R¹², N(R¹²)C(O)R^(12a), N(R¹²)S(O)₂R^(12a), N(R¹²)S(O)R^(12a),N(R¹²)C(O)OR^(12a), N(R¹²)C(O)N(R^(12a)R^(12b)), OC(O)N(R¹²R^(12a)), orC₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different; and wherein R¹¹, R^(11a),R¹², R^(12a), and R^(12b) are independently selected from the groupconsisting of: H and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different;wherein L² is a single chemical bond or a spacer; and wherein Z is acarrier group.
 2. The prodrug of claim 1; wherein X³ is O.
 3. Theprodrug of claim 1; wherein: X is N(R⁴); X¹ is C; and X³ is O.
 4. Theprodrug of claim 1; wherein X² is C(R⁷R^(7a)).
 5. The prodrug of claim1; wherein L¹ is selected from the group consisting of:

wherein: R is H, or C₁₋₄ alkyl; and Y is NH, O, or S.
 6. The prodrug ofclaim 1; wherein L¹ is selected from the group consisting of:

wherein R is H, or C₁₋₄ alkyl.
 7. The prodrug of claim 1; wherein: L² isa single chemical bond; or L²-Z is selected from the group consistingof: COOR⁹, OR⁹, C(O)R⁹, C(O)N(R⁹R^(9a)), S(O)₂N(R⁹R^(9a)),S(O)N(R⁹R^(9a)), S(O)₂R⁹, S(O)R⁹, N(R⁹)S(O)₂N(R^(9a)R^(9b)), SR⁹,N(R⁹R^(9a)), OC(O)R⁹, N(R⁹)C(O)R^(9a), N(R⁹)S(O)₂R^(9a),N(R⁹)S(O)R^(9a), N(R⁹)C(O)OR^(9a), N(R⁹)C(O)N(R^(9a)R^(9b)),OC(O)N(R⁹R^(9a)), T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl;wherein T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R¹⁰, which are the same or different; andwherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R¹¹)—, —S(O)₂N(R¹¹)—, —S(O)N(R¹¹)—,—S(O)₂—, —S(O)—, —N(R¹¹)S(O)₂N(R^(11a))—, —S—, —N(R¹¹)—, —OC(O)R¹¹,—N(R¹¹)C(O)—, —N(R¹¹)S(O)₂—, —N(R¹¹)S(O)—, —N(R¹¹)C(O)O—,—N(R¹¹)C(O)N(R^(11a))—, and —OC(O)N(R¹¹R^(11a)); and wherein: R⁹,R^(9a), and R^(9b) are independently selected from the group consistingof: H, Z, T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein T,C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally substitutedwith one or more R¹⁰, which are the same or different; and wherein C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted byone or more groups selected from the group consisting of: T, —C(O)O—,—O—, —C(O)—, —C(O)N(R¹¹)—, —S(O)₂N(R¹¹)—, —S(O)N(R¹¹)—, —S(O)₂—, —S(O)—,—N(R¹¹)S(O)₂N(R^(11a))—, —S—, —N(R¹¹)—, —OC(O)R¹¹, —N(R¹¹)C(O)—,—N(R¹¹)S(O)₂—, —N(R¹¹)S(O)—, —N(R¹¹)C(O)O—, —N(R¹¹)C(O)N(R^(11a))—, and—OC(O)N(R¹¹R^(11a)); T is selected from the group consisting of: phenyl,naphthyl, indenyl, indenyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4 to 7membered heterocyclyl, and 9 to 11 membered heterobicyclyl; wherein T isoptionally substituted with one or more R¹⁰, which are the same ordifferent; R¹⁰ is selected from the group consisting of: Z, halogen, CN,oxo (═O), COOR¹², OR¹², C(O)R¹², C(O)N(R¹²R^(12a)), S(O)₂N(R¹²R^(12a)),S(O)N(R¹²R^(12a)), S(O)₂R¹², S(O)R¹², N(R¹²)S(O)₂N(R^(12a)R^(12b)),SR¹², N(R¹², R^(12a)), NO₂; OC(O)R₁₂, N(R¹²)C(O)R^(12a),N(R¹²)S(O)₂R^(12a), N(R¹²)S(O)R^(12a), N(R¹²)C(O)OR^(12a),N(R¹²)C(O)N(R^(12a)R^(12b)), OC(O)N(R¹²R^(12a)), and C₁₋₆ alkyl; whereinC₁₋₆ alkyl is optionally substituted with one or more halogen, which arethe same or different; and R¹¹, R^(11a), R¹², R^(12a), and R^(12b) areindependently selected from the group consisting of: H, Z, or C₁₋₆alkyl; wherein C₁₋₆ alkyl is optionally substituted with one or morehalogen, which are the same or different; provided that one of R⁹,R^(9a), R^(9b), R¹⁰, R¹¹, R^(11a), R¹², R^(12a), and R^(12b) is Z. 8.The prodrug of claim 1; wherein L² is a C₁₋₂₀ alkyl chain, which is:optionally interrupted by one or more groups independently selected from—O— and C(O)N(R^(3aa)); and optionally substituted with one or moregroups independently selected from OH and C(O)N(R^(3aa)R^(aaa)); andwherein R^(3aa) and R^(3aaa) are independently selected from the groupconsisting of H, and C₁₋₄ alkyl.
 9. The prodrug of claim 1; wherein L²has a molecular weight in the range of from 14 g/mol to 750 g/mol. 10.The prodrug of claim 1; wherein L² is attached to Z via a terminal groupselected from the group consisting of:


11. The prodrug of claim 1; wherein L is represented by formula (Ia):

wherein R^(3aa) and R^(3aaa): are independently selected from the groupconsisting of H and C₁₋₄ alkyl; or are joined together with the nitrogenatom to which they are attached to form a 4 to 7 membered heterocycle.12. The prodrug of claim 1; wherein L is represented by formula (Ib):

wherein R^(3aa) is H or C₁₋₄ alkyl.
 13. The prodrug of claim 1; whereinR¹ in formula (I) is L²-Z.
 14. The prodrug of claim 1; wherein R³ informula (I) is L²-Z.
 15. The prodrug of claim 1; wherein R³ and R^(3a)in formula (I) are joined together with the nitrogen atom to which theyare attached to form a 4 to 7 membered heterocycle which is substitutedwith L²-Z.
 16. The prodrug of claim 1; wherein Z is a polymer of atleast 500 Da or a C₈₋₁₈ alkyl group.
 17. The prodrug of claim 1; whereinZ is selected from the group of optionally crosslinked polymersconsisting of: poly(propylene glycol), poly(ethylene glycol), dextran,chitosan, hyaluronic acid, alginate, xylan, mannan, carrageenan,agarose, cellulose, starch, hydroxyalkyl starch (HAS), poly(vinylalcohols), poly(oxazolines), poly(anhydrides), poly(ortho esters),poly(carbonates), poly(urethanes), poly(acrylic acids),poly(acrylamides), poly(acrylates), poly(methacrylates),poly(organophosphazenes), polyoxazoline, poly(siloxanes), poly(amides),poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters),poly(iminocarbonates), poly(amino acids), collagen, gelatin, hydrogel, ablood plasma protein, and copolymers thereof.
 18. The prodrug of claim1; wherein Z is a linear or branched poly(ethylene glycol) with amolecular weight from 2,000 Da to 150,000 Da.
 19. A pharmaceuticalcomposition comprising: a prodrug of claim 1 or a pharmaceutical saltthereof; and a pharmaceutically acceptable excipient.
 20. A methodcomprising: administering the prodrug of claim
 1. 21. A methodcomprising: administering the pharmaceutical composition of claim 19.